Serine dehydratase homolog

ABSTRACT

The invention provides a human serine dehydratase homolog (SDHH) and polynucleotides which identify and encode SDHH. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for diagnosing, treating or preventing disorders associated with expression of SDHH.

FIELD OF THE INVENTION

This invention relates to nucleic acid and amino acid sequences of aserine dehydratase homolog and to the use of these sequences in thediagnosis, treatment, and prevention of disorders of metabolism andcancer.

BACKGROUND OF THE INVENTION

Serine dehydratase (SDH) is an enzyme involved in gluconeogenesis, theformation of glucose, the primary fuel for cellular processes, fromamino acids and certain types of fat. Gluconeogenesis usually occurs inresponse to a decrease in supply, or increase in demand, for glucose.SDH converts serine to pyruvate and NH₄ ⁺, following a dehydration step.SDH competes with serine hydroxymethyltransferase and serineaminotransferase for available serine. (Snell et al. (1988) Br. J.Cancer 57:87-90.) SDH also catalyzes the deamination of L-threonine,preferring L-threonine at higher pH values and L-serine at lower pHvalues. (Pagani et al. 1989, Boll. Soc. Ital. Biol. Sper. 65: 625-629.)A variety of SDHs have been observed in organisms ranging from bacteriato vertebrates. A motif which interacts with SDH's pyridoxal5′-phosphate cofactor in several B6 enzymes is considered characteristicof SDH. (Noda et al. 1988, FEBS Lett. 234:331-335)

SDH is synthesized primarily in the liver. (Su et al. 1992, Gene120:301-306.) In rats, which are nocturnal feeders, SDH exhibits acircadian rhythm, reaching a maximum at the onset of darkness and aminimum at the onset of light. (Ogawa et al. 1995, Histochem. J.27:380-387.) Variation in SDH levels appears to be generated at thelevel of transcription. (Ogawa et al. 1994, Arch. Biochem. Biophys.308:285-291.) Cis-acting DNA elements required for liver-specificexpression of the SDH gene have been identified. Expression of SDH mRNAin cultured hepatocytes appears to be regulated in G0/G1 transitionbefore entry into the S phase of the cell cycle. (Noda et al., 1990,Biochem. Biophys. Res. Commun. 168:335-342.)

Gluconeogenesis is regulated by a variety of hormones responsive to suchfactors as age, diet, and stress. Acute hormonal regulation of livercarbohydrate metabolism mainly involves changes in cytosolic levels ofcyclic adenosine monophosphate (cAMP) and Ca++(Exton, 1987, DiabetesMetab. Rev. 3:163-183). Epinephrine and glucagon both stimulategluconeogenesis by activating adenylate cyclase in the liver plasmamembrane resulting in accumulation of cAMP. cAMP up-regulates SDHtranscription.

Induction of translatable mRNA for SDH in primary cultured rathepatocytes requires both dexamethasone and glucagon or cAMP, a uniquehormone requirement. Insulin and catecholamine are antagonists of SDHinduction (Ichihara et al. 1982, Mol. Cell Biochem. 43:145-160.) Theseeffects are mediated by the alpha-I adrenergic signal transfer system.The dexamethasone induction is age-dependent, apparently in correlationto the degree of methylation of the promoter region of the gene. (Bohmeet al., 1987, Adv. Enzyme Regul. 26:31-61.) SDH transcription is inducedin rats near birth, when their diet changes from a continuous supply ofglucose via placental blood to relatively fat-rich carbohydrate-poorblood. (Bohme et al 1983 Experientia 39:473-483.)

A number of conditions and disorders involve SDH, including metabolicdisorders and cancer. Gluconeogenesis from amino acids is enhanced afteracute renal failure. Nephrectomized rats show significantly elevated SDHactivity. Serine may play a special role as a substrate forgluconeogenesis in acute uremic rats, probably mediated by an activationof SDH. (Frohlich et al., 1977, Eur. J. Clin. Invest. 7:261-268.)Attempts have been made to develop procedures for estimation of SDHactivity in blood serum to aid in detection of a variety of liver tissueimpairments. (Muzhichenko et al. 1981, Vopr. Med. Khim 27:408-412.)Obese Zucker rats show significantly depressed hepatic SDH activity, andthe activity does not increase in response to starvation as in leanrats. (Domenech et al. 1993, Cell. Mol. Biol. (Noisy-le-grand)39:405-414.) SDH mRNA levels are markedly increased instreptozotocin-induced diabetes. (Ogawa, supra.) Neonatal insulinresistance which contributes to neonatal hyperglycemia has been linkedto epinephrine counteracting insulin's ability to decrease SDH genetranscription. (Feng et al., 1996, Biochem. Mol. Med. 57:91-96.)

The balance of SDH, serine hydroxymethyltransferase and serineaminotransferase activities is altered in human colon carcinoma and ratsarcoma. SDH and serine aminotransferase activities are absent in humancolon carcinoma and rat sarcoma, while the activity of serinehydroxymethyltransferase is markedly increased. This change may besymptomatic of the biochemical commitment to cellular replication incancer cells. Snell et al. (supra.)

The discovery of a new serine dehydratase homolog and thepolynucleotides encoding it satisfies a need in the art by providing newcompositions which are useful in the diagnosis, treatment, andprevention of disorders of metabolism and cancer.

SUMMARY OF THE INVENTION

The invention is based on the discovery of a new human serinedehydratase homolog (SDHH), the polynucleotides encoding SDHH, and theuse of these compositions for the diagnosis, treatment, or prevention ofdisorders of metabolism and cancer.

The invention features a substantially purified polypeptide comprisingthe amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.

The invention further provides a substantially purified variant havingat least 90% amino acid sequence identity to the amino acid sequence ofSEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention also provides anisolated and purified polynucleotide encoding the polypeptide comprisingthe sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The inventionalso includes an isolated and purified polynucleotide variant having atleast 90% polynucleotide sequence identity to the polynucleotideencoding the polypeptide comprising the amino acid sequence of SEQ IDNO:1 or a fragment of SEQ ID NO:1.

The invention further provides an isolated and purified polynucleotidewhich hybridizes under stringent conditions to the polynucleotideencoding the polypeptide comprising the amino acid sequence of SEQ IDNO:1 or a fragment of SEQ ID NO:1, as well as an isolated and purifiedpolynucleotide which is complementary to the polynucleotide encoding thepolypeptide comprising the amino acid sequence of SEQ ID NO:1 or afragment of SEQ ID NO:1.

The invention also provides an isolated and purified polynucleotidecomprising the polynucleotide sequence of SEQ ID NO:2 or a fragment ofSEQ ID NO:2, and an isolated and purified polynucleotide variant havingat least 90% polynucleotide sequence identity to the polynucleotidecomprising the polynucleotide sequence of SEQ ID NO:2 or a fragment ofSEQ ID NO:2. The invention also provides an isolated and purifiedpolynucleotide having a sequence complementary to the polynucleotidecomprising the polynucleotide sequence of SEQ ID NO:2 or a fragment ofSEQ ID NO:2.

The invention further provides an expression vector comprising at leasta fragment of the polynucleotide encoding the polypeptide comprising thesequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. In another aspect,the expression vector is contained within a host cell.

The invention also provides a method for producing a polypeptidecomprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQID NO:1, the method comprising the steps of: (a) culturing the host cellcomprising an expression vector containing at least a fragment of apolynucleotide encoding the polypeptide comprising the amino acidsequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 under conditionssuitable for the expression of the polypeptide; and (b) recovering thepolypeptide from the host cell culture.

The invention also provides a pharmaceutical composition comprising asubstantially purified polypeptide having the sequence of SEQ ID NO:1 ora fragment of SEQ ID NO:1 in conjunction with a suitable pharmaceuticalcarrier.

The invention further includes a purified antibody which binds to apolypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQID NO:1, as well as a purified agonist and a purified antagonist of thepolypeptide.

The invention also provides a method for treating or preventing adisorder of metabolism associated with decreased expression of SDHH, themethod comprising administering to a subject in need of such treatmentan effective amount of a pharmaceutical composition comprisingsubstantially purified polypeptide having the amino acid sequence of SEQID NO:1 or a fragment of SEQ ID NO:1.

The invention also provides a method for treating or preventing adisorder of metabolism associated with increased expression of SDHH, themethod comprising administering to a subject in need of such treatmentan effective amount of an antagonist of the polypeptide having the aminoacid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.

The invention also provides a method for treating or preventing acancer, the method comprising administering to a subject in need of suchtreatment an effective amount of a pharmaceutical composition comprisingsubstantially purified polypeptide having the amino acid sequence of SEQID NO:1 or a fragment of SEQ ID NO:1.

The invention also provides a method for detecting a polynucleotideencoding a polypeptide comprising the amino acid sequence of SEQ ID NO:1or a fragment of SEQ ID NO:1 in a biological sample containing nucleicacids, the method comprising the steps of: (a) hybridizing thecomplement of the polynucleotide encoding the polypeptide comprising theamino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 to atleast one of the nucleic acids of the biological sample, thereby forminga hybridization complex; and (b) detecting the hybridization complex,wherein the presence of the hybridization complex correlates with thepresence of a polynucleotide encoding the polypeptide comprising theamino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 in thebiological sample. In one aspect, the nucleic acids of the biologicalsample are amplified by the polymerase chain reaction prior to thehybridizing step.

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleotide sequences, and methods aredescribed, it is understood that this invention is not limited to theparticular methodology, protocols, cell lines, vectors, and reagentsdescribed, as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, a reference to “ahost cell” includes a plurality of such host cells, and a reference to“an antibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are now described. All publications mentionedherein are cited for the purpose of describing and disclosing the celllines, vectors, and methodologies which are reported in the publicationsand which might be used in connection with the invention. Nothing hereinis to be construed as an admission that the invention is not entitled toantedate such disclosure by virtue of prior invention.

DEFINITIONS

“SDHH,” as used herein, refers to the amino acid sequences ofsubstantially purified SDHH obtained from any species, particularly amammalian species, including bovine, ovine, porcine, murine, equine, andpreferably the human species, from any source, whether natural,synthetic, semi-synthetic, or recombinant.

The term “agonist,” as used herein, refers to a molecule which, whenbound to SDHH, increases or prolongs the duration of the effect of SDHH.Agonists may include proteins, nucleic acids, carbohydrates, or anyother molecules which bind to and modulate the effect of SDHH.

An “allelic variant,” as this term is used herein, is an alternativeform of the gene encoding SDHH. Allelic variants may result from atleast one mutation in the nucleic acid sequence and may result inaltered mRNAs or in polypeptides whose structure or function may or maynot be altered. Any given natural or recombinant gene may have none,one, or many allelic forms. Common mutational changes which give rise toallelic variants are generally ascribed to natural deletions, additions,or substitutions of nucleotides. Each of these types of changes mayoccur alone, or in combination with the others, one or more times in agiven sequence.

“Altered” nucleic acid sequences encoding SDHH, as described herein,include those sequences with deletions, insertions, or substitutions ofdifferent nucleotides, resulting in a polynucleotide the same as SDHH ora polypeptide with at least one functional characteristic of SDHH.Included within this definition are polymorphisms which may or may notbe readily detectable using a particular oligonucleotide probe of thepolynucleotide encoding SDHH, and improper or unexpected hybridizationto allelic variants, with a locus other than the normal chromosomallocus for the polynucleotide sequence encoding SDHH. The encoded proteinmay also be “altered,” and may contain deletions, insertions, orsubstitutions of amino acid residues which produce a silent change andresult in a functionally equivalent SDHH. Deliberate amino acidsubstitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues, as long as the biological orimmunological activity of SDHH is retained. For example, negativelycharged amino acids may include aspartic acid and glutamic acid,positively charged amino acids may include lysine and arginine, andamino acids with uncharged polar head groups having similarhydrophilicity values may include leucine, isoleucine, and valine;glycine and alanine; asparagine and glutamine; serine and threonine; andphenylalanine and tyrosine.

The terms “amino acid” or “amino acid sequence,” as used herein, referto an oligopeptide, peptide, polypeptide, or protein sequence, or afragment of any of these, and to naturally occurring or syntheticmolecules. In this context, “fragments,” “immunogenic fragments,” or“antigenic fragments” refer to fragments of SDHH which are preferablyabout 5 to about 15 amino acids in length, most preferably 14 aminoacids, and which retain some biological activity or immunologicalactivity of SDHH. Where “amino acid sequence” is recited herein to referto an amino acid sequence of a naturally occurring protein molecule,“amino acid sequence” and like terms are not meant to limit the aminoacid sequence to the complete native amino acid sequence associated withthe recited protein molecule.

“Amplification,” as used herein, relates to the production of additionalcopies of a nucleic acid sequence. Amplification is generally carriedout using polymerase chain reaction (PCR) technologies well known in theart. (See, e.g., Dieffenbach, C. W. and G. S. Dveksler (1995) PCRPrimer, a Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.,pp. 1-5.)

The term “antagonist,” as it is used herein, refers to a molecule which,when bound to SDHH, decreases the amount or the duration of the effectof the biological or immunological activity of SDHH. Antagonists mayinclude proteins, nucleic acids, carbohydrates, antibodies, or any othermolecules which decrease the effect of SDHH.

As used herein, the term “antibody” refers to intact molecules as wellas to fragments thereof, such as Fab, F(ab′)₂, and Fv fragments, whichare capable of binding the epitopic determinant. Antibodies that bindSDHH polypeptides can be prepared using intact polypeptides or usingfragments containing small peptides of interest as the immunizingantigen. The polypeptide or oligopeptide used to immunize an animal(e.g., a mouse, a rat, or a rabbit) can be derived from the translationof RNA, or synthesized chemically, and can be conjugated to a carrierprotein if desired. Commonly used carriers that are chemically coupledto peptides include bovine serum albumin, thyroglobulin, and keyholelimpet hemocyanin (KLH). The coupled peptide is then used to immunizethe animal.

The term “antigenic determinant,” as used herein, refers to thatfragment of a molecule (i.e., an epitope) that makes contact with aparticular antibody. When a protein or a fragment of a protein is usedto immunize a host animal, numerous regions of the protein may inducethe production of antibodies which bind specifically to antigenicdeterminants (given regions or three-dimensional structures on theprotein). An antigenic determinant may compete with the intact antigen(i.e., the immunogen used to elicit the immune response) for binding toan antibody.

The term “antisense,” as used herein, refers to any compositioncontaining a nucleic acid sequence which is complementary to the “sense”strand of a specific nucleic acid sequence. Antisense molecules may beproduced by any method including synthesis or transcription. Onceintroduced into a cell, the complementary nucleotides combine withnatural sequences produced by the cell to form duplexes and to blockeither transcription or translation. The designation “negative” canrefer to the antisense strand, and the designation “positive” can referto the sense strand.

As used herein, the term “biologically active,” refers to a proteinhaving structural, regulatory, or biochemical functions of a naturallyoccurring molecule. Likewise, “immunologically active” refers to thecapability of the natural, recombinant, or synthetic SDHH, or of anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

The terms “complementary” or “complementarity,” as used herein, refer tothe natural binding of polynucleotides under permissive salt andtemperature conditions by base pairing. For example, the sequence“A-G-T” binds to the complementary sequence “T-C-A.” Complementaritybetween two single-stranded molecules may be “partial,” such that onlysome of the nucleic acids bind, or it may be “complete,” such that totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of the hybridization between the nucleicacid strands. This is of particular importance in amplificationreactions, which depend upon binding between nucleic acids strands, andin the design and use of peptide nucleic acid (PNA) molecules.

A “composition comprising a given polynucleotide sequence” or a“composition comprising a given amino acid sequence,” as these terms areused herein, refer broadly to any composition containing the givenpolynucleotide or amino acid sequence. The composition may comprise adry formulation, an aqueous solution, or a sterile composition.Compositions comprising polynucleotide sequences encoding SDHH orfragments of SDHH may be employed as hybridization probes. The probesmay be stored in freeze-dried form and may be associated with astabilizing agent such as a carbohydrate. In hybridizations, the probemay be deployed in an aqueous solution containing salts, e.g., NaCl,detergents, e.g.,sodium dodecyl sulfate (SDS), and other components,e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.

“Consensus sequence,” as used herein, refers to a nucleic acid sequencewhich has been resequenced to resolve uncalled bases, extended usingXL-PCR™ (Perkin Elmer, Norwalk, Conn.) in the 5′ and/or the 3′direction, and resequenced, or which has been assembled from theoverlapping sequences of more than one Incyte Clone using a computerprogram for fragment assembly, such as the GELVIEW™ Fragment Assemblysystem (GCG, Madison, Wis.). Some sequences have been both extended andassembled to produce the consensus sequence.

As used herein, the term “correlates with expression of apolynucleotide” indicates that the detection of the presence of nucleicacids, the same or related to a nucleic acid sequence encoding SDHH, byNorthern analysis is indicative of the presence of nucleic acidsencoding SDHH in a sample, and thereby correlates with expression of thetranscript from the polynucleotide encoding SDHH.

A “deletion,” as the term is used herein, refers to a change in theamino acid or nucleotide sequence that results in the absence of one ormore amino acid residues or nucleotides.

The term “derivative,” as used herein, refers to the chemicalmodification of a polypeptide sequence, or a polynucleotide sequence.Chemical modifications of a polynucleotide sequence can include, forexample, replacement of hydrogen by an alkyl, acyl, or amino group. Aderivative polynucleotide encodes a polypeptide which retains at leastone biological or immunological function of the natural molecule. Aderivative polypeptide is one modified by glycosylation, pegylation, orany similar process that retains at least one biological orimmunological function of the polypeptide from which it was derived.

The term “similarity,” as used herein, refers to a degree ofcomplementarity. There may be partial similarity or complete similarity.The word “identity” may substitute for the word “similarity.” Apartially complementary sequence that at least partially inhibits anidentical sequence from hybridizing to a target nucleic acid is referredto as “substantially similar.” The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or Northern blot, solutionhybridization, and the like) under conditions of reduced stringency. Asubstantially similar sequence or hybridization probe will compete forand inhibit the binding of a completely similar (identical) sequence tothe target sequence under conditions of reduced stringency. This is notto say that conditions of reduced stringency are such that non-specificbinding is permitted, as reduced stringency conditions require that thebinding of two sequences to one another be a specific (i.e., aselective) interaction. The absence of non-specific binding may betested by the use of a second target sequence which lacks even a partialdegree of complementarity (e.g., less than about 30% similarity oridentity). In the absence of non-specific binding, the substantiallysimilar sequence or probe will not hybridize to the secondnon-complementary target sequence.

The phrases “percent identity” or “% identity” refer to the percentageof sequence similarity found in a comparison of two or more amino acidor nucleic acid sequences. Percent identity can be determinedelectronically, e.g., by using the MegAlign™ program (DNASTAR, Inc.,Madison Wis.). The MegAlign™ program can create alignments between twoor more sequences according to different methods, e.g., the clustalmethod. (See, e.g., Higgins, D. G. and P. M. Sharp (1988) Gene73:237-244.) The clustal algorithm groups sequences into clusters byexamining the distances between all pairs. The clusters are alignedpairwise and then in groups. The percentage similarity between two aminoacid sequences, e.g., sequence A and sequence B, is calculated bydividing the length of sequence A, minus the number of gap residues insequence A, minus the number of gap residues in sequence B, into the sumof the residue matches between sequence A and sequence B, times onehundred. Gaps of low or of no similarity between the two amino acidsequences are not included in determining percentage similarity. Percentidentity between nucleic acid sequences can also be counted orcalculated by other methods known in the art, e.g., the Jotun Heinmethod. (See, e.g., Hein, J. (1990) Methods Enzymol. 183:626-645.)Identity between sequences can also be determined by other methods knownin the art, e.g., by varying hybridization conditions.

“Human artificial chromosomes” (HACs), as described herein, are linearmicrochromosomes which may contain DNA sequences of about 6 kb to 10 Mbin size, and which contain all of the elements required for stablemitotic chromosome segregation and maintenance. (See, e.g., Harrington,J. J. et al. (1997) Nat Genet. 15:345-355.)

The term “humanized antibody,” as used herein, refers to antibodymolecules in which the amino acid sequence in the non-antigen bindingregions has been altered so that the antibody more closely resembles ahuman antibody, and still retains its original binding ability.

“Hybridization,” as the term is used herein, refers to any process bywhich a strand of nucleic acid binds with a complementary strand throughbase pairing.

As used herein, the term “hybridization complex” refers to a complexformed between two nucleic acid sequences by virtue of the formation ofhydrogen bonds between complementary bases. A hybridization complex maybe formed in solution (e.g., C₀t or R₀t analysis) or formed between onenucleic acid sequence present in solution and another nucleic acidsequence immobilized on a solid support (e.g., paper, membranes,filters, chips, pins or glass slides, or any other appropriate substrateto which cells or their nucleic acids have been fixed).

The words “insertion” or “addition,” as used herein, refer to changes inan amino acid or nucleotide sequence resulting in the addition of one ormore amino acid residues or nucleotides, respectively, to the sequencefound in the naturally occurring molecule.

“Immune response” can refer to conditions associated with inflammation,trauma, immune disorders, or infectious or genetic disease, etc. Theseconditions can be characterized by expression of various factors, e.g.,cytokines, chemokines, and other signaling molecules, which may affectcellular and systemic defense systems.

The term “microarray,” as used herein, refers to an arrangement ofdistinct polynucleotides arrayed on a substrate, e.g., paper, nylon orany other type of membrane, filter, chip, glass slide, or any othersuitable solid support.

The terms “element” or “array element” as used herein in a microarraycontext, refer to hybridizable polynucleotides arranged on the surfaceof a substrate.

The term “modulate,” as it appears herein, refers to a change in theactivity of SDHH. For example, modulation may cause an increase or adecrease in protein activity, binding characteristics, or any otherbiological, functional, or immunological properties of SDHH.

The phrases “nucleic acid” or “nucleic acid sequence,” as used herein,refer to a nucleotide, oligonucleotide, polynucleotide, or any fragmentthereof. These phrases also refer to DNA or RNA of genomic or syntheticorigin which may be single-stranded or double-stranded and may representthe sense or the antisense strand, to peptide nucleic acid (PNA), or toany DNA-like or RNA-like material. In this context, “fragments” refersto those nucleic acid sequences which, when translated, would producepolypeptides retaining some functional characteristic, e.g.,antigenicity, or structural domain characteristic, e.g., ATP-bindingsite, of the full-length polypeptide.

The terms “operably associated” or “operably linked,” as used herein,refer to functionally related nucleic acid sequences. A promoter isoperably associated or operably linked with a coding sequence if thepromoter controls the translation of the encoded polypeptide. Whileoperably associated or operably linked nucleic acid sequences can becontiguous and in the same reading frame, certain genetic elements,e.g., repressor genes, are not contiguously linked to the sequenceencoding the polypeptide but still bind to operator sequences thatcontrol expression of the polypeptide.

The term “oligonucleotide,” as used herein, refers to a nucleic acidsequence of at least about 6 nucleotides to 60 nucleotides, preferablyabout 15 to 30 nucleotides, and most preferably about 20 to 25nucleotides, which can be used in PCR amplification or in ahybridization assay or microarray. As used herein, the term“oligonucleotide” is substantially equivalent to the terms “amplimer,”“primer,” “oligomer,” and “probe,” as these terms are commonly definedin the art.

“Peptide nucleic acid” (PNA), as used herein, refers to an antisensemolecule or anti-gene agent which comprises an oligonucleotide of atleast about 5 nucleotides in length linked to a peptide backbone ofamino acid residues ending in lysine. The terminal lysine conferssolubility to the composition. PNAs preferentially bind complementarysingle stranded DNA or RNA and stop transcript elongation, and may bepegylated to extend their lifespan in the cell. (See, e.g., Nielsen, P.E. et al. (1993) Anticancer Drug Des. 8:53-63.)

The term “sample,” as used herein, is used in its broadest sense. Abiological sample suspected of containing nucleic acids encoding SDHH,or fragments thereof, or SDHH itself, may comprise a bodily fluid; anextract from a cell, chromosome, organelle, or membrane isolated from acell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a solidsupport; a tissue; a tissue print; etc.

As used herein, the terms “specific binding” or “specifically binding”refer to that interaction between a protein or peptide and an agonist,an antibody, or an antagonist. The interaction is dependent upon thepresence of a particular structure of the protein, e.g., the antigenicdeterminant or epitope, recognized by the binding molecule. For example,if an antibody is specific for epitope “A,” the presence of apolypeptide containing the epitope A, or the presence of free unlabeledA, in a reaction containing free labeled A and the antibody will reducethe amount of labeled A that binds to the antibody.

As used herein, the term “stringent conditions” refers to conditionswhich permit hybridization between polynucleotides and the claimedpolynucleotides. Stringent conditions can be defined by saltconcentration, the concentration of organic solvent (e.g., formamide),temperature, and other conditions well known in the art. In particular,stringency can be increased by reducing the concentration of salt,increasing the concentration of formamide, or raising the hybridizationtemperature.

For example, stringent salt concentration will ordinarily be less thanabout 750 mM NaCl and 75 mM trisodium citrate, preferably less thanabout 500 mM NaCl and 50 mM trisodium citrate, and most preferably lessthan about 250 mM NaCl and 25 mM trisodium citrate. Low stringencyhybridization can be obtained in the absence of organic solvent, e.g.,formamide, while high stringency hybridization can be obtained in thepresence of at least about 35% formamide, and most preferably at leastabout 50% formamide. Stringent temperature conditions will ordinarilyinclude temperatures of at least about 30° C., more preferably of atleast about 37° C., and most preferably of at least about 42° C. Varyingadditional parameters, such as hybridization time, the concentration ofdetergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion orexclusion of carrier DNA, are well known to those skilled in the art.Various levels of stringency are accomplished by combining these variousconditions as needed. In a preferred embodiment, hybridization willoccur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. Ina more preferred embodiment, hybridization will occur at 37° C. in 500mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg/mldenatured salmon sperm DNA (ssDNA). In a most preferred embodiment,hybridization will occur at 42° C. in 250 mM NaCl, 25 mM trisodiumcitrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variationson these conditions will be readily apparent to those skilled in theart.

The washing steps which follow hybridization can also vary instringency. Wash stringency conditions can be defined by saltconcentration and by temperature. As above, wash stringency can beincreased by decreasing salt concentration or by increasing temperature.For example, stringent salt concentration for the wash steps willpreferably be less than about 30 mM NaCl and 3 mM trisodium citrate, andmost preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.Stringent temperature conditions for the wash steps will ordinarilyinclude temperature of at least about 25° C., more preferably of atleast about 42° C., and most preferably of at least about 68° C. In apreferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, washsteps will occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and0.1% SDS. In a most preferred embodiment, wash steps will occur at 68°C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additionalvariations on these conditions will be readily apparent to those skilledin the art.

The term “substantially purified,” as used herein, refers to nucleicacid or amino acid sequences that are removed from their naturalenvironment and are isolated or separated, and are at least about 60%free, preferably about 75% free, and most preferably about 90% free fromother components with which they are naturally associated.

A “substitution,” as used herein, refers to the replacement of one ormore amino acids or nucleotides by different amino acids or nucleotides,respectively.

“Transformation,” as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. Transformation mayoccur under natural or artificial conditions according to variousmethods well known in the art, and may rely on any known method for theinsertion of foreign nucleic acid sequences into a prokaryotic oreukaryotic host cell. The method for transformation is selected based onthe type of host cell being transformed and may include, but is notlimited to, viral infection, electroporation, heat shock, lipofection,and particle bombardment. The term “transformed” cells includes stablytransformed cells in which the inserted DNA is capable of replicationeither as an autonomously replicating plasmid or as part of the hostchromosome, as well as transiently transformed cells which express theinserted DNA or RNA for limited periods of time.

A “variant” of SDHH, as used herein, refers to an amino acid sequencethat is altered by one or more amino acids. The variant may have“conservative” changes, wherein a substituted amino acid has similarstructural or chemical properties (e.g., replacement of leucine withisoleucine). More rarely, a variant may have “nonconservative” changes(e.g., replacement of glycine with tryptophan). Analogous minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, LASERGENE™ software.

The Invention

The invention is based on the discovery of a new human serinedehydratase homolog (SDHH), the polynucleotides encoding SDHH, and theuse of these compositions for the diagnosis, treatment, or prevention ofdisorders of metabolism and cancer.

Nucleic acids encoding the SDHH of the present invention were firstidentified in Incyte Clone 2752518 from the THP-1 promonocyte cell cDNAlibrary (THP1AZS08) using a computer search, e.g., BLAST, for amino acidsequence alignments. A consensus sequence, SEQ ID NO:2, was derived fromthe following overlapping and/or extended nucleic acid sequences: IncyteClones 638642 (BRSTNOT03), 823439 (KERANOT02) and 2752518 (THP1AZS08).

In one embodiment, the invention encompasses a polypeptide comprisingthe amino acid sequence of SEQ ID NO:1. SDHH is 329 amino acids inlength and has a potential casein kinase II phosphorylation site atresidue T218; and two potential protein kinase C phosphorylation sitesat residues S46 and T116. SDHH has the serine/threonine dehydratasepyridoxal-phosphate attachment site at E39. SDHH has chemical andstructural similarity with rat liver serine dehydratase (GI 57225; SEQID NO:3), and human liver serine dehydratase (GI 338030; SEQ ID NO:4) Inparticular, SDHH and rat liver serine dehydratase share 53.2% identity,and SDHH and human liver serine dehydratase share 56.7% identity. Aregion of unique sequence in SDHH from about amino acid 2 to about aminoacid 8 is encoded by a fragment of SEQ ID NO:2 from about nucleotide 300to about nucleotide 318. Northern analysis shows the expression of SDHHin various libraries, 48% of which are cancerous, 29% are involved inimmune response, and 23% are fetal, cell line or proliferating, 22% arefrom gastrointestinal tissue, 16% from immune tissue, 16% fromreproductive tissue, and 12% are from cardiovascular tissue.

The invention also encompasses SDHH variants. A preferred SDHH variantis one which has at least about 80%, more preferably at least about 90%,and most preferably at least about 95% amino acid sequence identity tothe SDHH amino acid sequence, and which contains at least one functionalor structural characteristic of SDHH.

The invention also encompasses polynucleotides which encode SDHH. In aparticular embodiment, the invention encompasses a polynucleotidesequence comprising the sequence of SEQ ID NO:2, which encodes an SDHH.

The invention also encompasses a variant of a polynucleotide sequenceencoding SDHH. In particular, such a variant polynucleotide sequencewill have at least about 80%, more preferably at least about 90%, andmost preferably at least about 95% polynucleotide sequence identity tothe polynucleotide sequence encoding SDHH. A particular aspect of theinvention encompasses a variant of SEQ ID NO:2 which has at least about80%, more preferably at least about 90%, and most preferably at leastabout 95% polynucleotide sequence identity to SEQ ID NO:2. Any one ofthe polynucleotide variants described above can encode an amino acidsequence which contains at least one functional or structuralcharacteristic of SDHH.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of polynucleotidesequences encoding SDHH, some bearing minimal similarity to thepolynucleotide sequences of any known and naturally occurring gene, maybe produced. Thus, the invention contemplates each and every possiblevariation of polynucleotide sequence that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet genetic code as applied tothe polynucleotide sequence of naturally occurring SDHH, and all suchvariations are to be considered as being specifically disclosed.

Although nucleotide sequences which encode SDHH and its variants arepreferably capable of hybridizing to the nucleotide sequence of thenaturally occurring SDHH under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding SDHH or its derivatives possessing a substantially differentcodon usage, e.g., inclusion of non-naturally occurring codons. Codonsmay be selected to increase the rate at which expression of the peptideoccurs in a particular prokaryotic or eukaryotic host in accordance withthe frequency with which particular codons are utilized by the host.Other reasons for substantially altering the nucleotide sequenceencoding SDHH and its derivatives without altering the encoded aminoacid sequences include the production of RNA transcripts having moredesirable properties, such as a greater half-life, than transcriptsproduced from the naturally occurring sequence.

The invention also encompasses production of DNA sequences which encodeSDHH and SDHH derivatives, or fragments thereof, entirely by syntheticchemistry. After production, the synthetic sequence may be inserted intoany of the many available expression vectors and cell systems usingreagents well known in the art. Moreover, synthetic chemistry may beused to introduce mutations into a sequence encoding SDHH or anyfragment thereof.

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridizing to the claimed polynucleotide sequences, and, inparticular, to those shown in SEQ ID NO:2, or a fragment of SEQ ID NO:2,under various conditions of stringency. (See, e.g., Wahl, G. M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A. R. (1987)Methods Enzymol. 152:507-511.)

Methods for DNA sequencing are well known and generally available in theart and may be used to practice any of the embodiments of the invention.The methods may employ such enzymes as the Klenow fragment of DNApolymerase I, Sequenase® (US Biochemical Corp., Cleveland, Ohio), Taqpolymerase (Perkin Elmer), thermostable T7 polymerase (Amersham,Chicago, Ill.), or combinations of polymerases and proofreadingexonucleases such as those found in the ELONGASE™ Amplification System(GIBCO BRL, Gaithersburg, Md.). Preferably, the process is automatedwith machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno,Nev.), Peltier Thermal Cycler (PTC200; MJ Research, Watertown, Mass.)and the ABI Catalyst and 373 and 377 DNA Sequencers (Perkin Elmer).

The nucleic acid sequences encoding SDHH may be extended utilizing apartial nucleotide sequence and employing various PCR-based methodsknown in the art to detect upstream sequences, such as promoters andregulatory elements. For example, one method which may be employed,restriction-site PCR, uses universal and nested primers to amplifyunknown sequence from genomic DNA within a cloning vector. (See, e.g.,Sarkar, G. (1993) PCR Methods Applic. 2:318-322.) Another method,inverse PCR, uses primers that extend in divergent directions to amplifyunknown sequence from a circularized template. The template is derivedfrom restriction fragments comprising a known genomic locus andsurrounding sequences. (See, e.g., Triglia, T. et al. (1988) NucleicAcids Res. 16:8186.) A third method, capture PCR, involves PCRamplification of DNA fragments adjacent to known sequences in human andyeast artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al.(1991) PCR Methods Applic. 1:111-119.) In this method, multiplerestriction enzyme digestions and ligations may be used to insert anengineered double-stranded sequence into a region of unknown sequencebefore performing PCR. Other methods which may be used to retrieveunknown sequences are known in the art. (See, e.g., Parker, J. D. et al.(1991) Nucleic Acids Res. 19:3055-306). Additionally, one may use PCR,nested primers, and PromoterFinder™ libraries to walk genomic DNA(Clontech, Palo Alto, Calif.). This procedure avoids the need to screenlibraries and is useful in finding intron/exon junctions. For allPCR-based methods, primers may be designed using commercially availablesoftware, such as OLIGO™ 4.06 Primer Analysis software (NationalBiosciences Inc., Plymouth, Minn.) or another appropriate program, to beabout 22 to 30 nucleotides in length, to have a GC content of about 50%or more, and to anneal to the template at temperatures of about 68° C.to 72° C.

When screening for full-length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. In addition,random-primed libraries, which often include sequences containing the 5′regions of genes, are preferable for situations in which an oligo d(T)library does not yield a full-length cDNA. Genomic libraries may beuseful for extension of sequence into 5′ non-transcribed regulatoryregions.

Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentnucleotide-specific, laser-stimulated fluorescent dyes, and a chargecoupled device camera for detection of the emitted wavelengths.Output/light intensity may be converted to electrical signal usingappropriate software (e.g., Genotyper™ and Sequence Navigator™, PerkinElmer), and the entire process from loading of samples to computeranalysis and electronic data display may be computer controlled.Capillary electrophoresis is especially preferable for sequencing smallDNA fragments which may be present in limited amounts in a particularsample.

In another embodiment of the invention, polynucleotide sequences orfragments thereof which encode SDHH may be cloned in recombinant DNAmolecules that direct expression of SDHH, or fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced and used to express SDHH.

The nucleotide sequences of the present invention can be engineeredusing methods generally known in the art in order to alter SDHH-encodingsequences for a variety of purposes including, but not limited to,modification of the cloning, processing, and/or expression of the geneproduct. DNA shuffling by random fragmentation and PCR reassembly ofgene fragments and synthetic oligonucleotides may be used to engineerthe nucleotide sequences. For example, oligonucleotide-mediatedsite-directed mutagenesis may be used to introduce mutations that createnew restriction sites, alter glycosylation patterns, change codonpreference, produce splice variants, and so forth.

In another embodiment, sequences encoding SDHH may be synthesized, inwhole or in part, using chemical methods well known in the art. (See,e.g., Caruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser.215-223, and Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser.225-232.) Alternatively, SDHH itself or a fragment thereof may besynthesized using chemical methods. For example, peptide synthesis canbe performed using various solid-phase techniques. (See, e.g., Roberge,J. Y. et al. (1995) Science 269:202-204.) Automated synthesis may beachieved using the ABI 431A Peptide Synthesizer (Perkin Elmer).Additionally, the amino acid sequence of SDHH, or any part thereof, maybe altered during direct synthesis and/or combined with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

The peptide may be substantially purified by preparative highperformance liquid chromatography. (See, e.g, Chiez, R. M. and F. Z.Regnier (1990) Methods Enzymol. 182:392-421.) The composition of thesynthetic peptides may be confirmed by amino acid analysis or bysequencing. (See, e.g., Creighton, T. (1984) Proteins, Structures andMolecular Properties, WH Freeman and Co., New York, N.Y.)

In order to express a biologically active SDHH, the nucleotide sequencesencoding SDHH or derivatives thereof may be inserted into an appropriateexpression vector, i.e., a vector which contains the necessary elementsfor transcriptional and translational control of the inserted codingsequence in a suitable host. These elements include regulatorysequences, such as enhancers, constitutive and inducible promoters, and5′ and 3′ untranslated regions in the vector and in polynucleotidesequences encoding SDHH. Such elements may vary in their strength andspecificity. Specific initiation signals may also be used to achievemore efficient translation of sequences encoding SDHH. Such signalsinclude the ATG initiation codon and adjacent sequences, e.g. the Kozaksequence. In cases where sequences encoding SDHH and its initiationcodon and upstream regulatory sequences are inserted into theappropriate expression vector, no additional transcriptional ortranslational control signals may be needed. However, in cases whereonly coding sequence, or a fragment thereof, is inserted, exogenoustranslational control signals including an in-frame ATG initiation codonshould be provided by the vector. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers appropriate for the particular host cell system used. (See,e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.)

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing sequences encoding SDHH andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. (See, e.g., Sambrook, J.et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring HarborPress, Plainview, N.Y., ch. 4, 8, and 16-17; and Ausubel, F. M. et al.(1995, and periodic supplements) Current Protocols in Molecular Biology,John Wiley & Sons, New York, N.Y., ch. 9, 13, and 16.)

A variety of expression vector/host systems may be utilized to containand express sequences encoding SDHH. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith viral expression vectors (e.g., baculovirus); plant cell systemstransformed with viral expression vectors (e.g., cauliflower mosaicvirus (CaMV) or tobacco mosaic virus (TMV)) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Theinvention is not limited by the host cell employed.

In bacterial systems, a number of cloning and expression vectors may beselected depending upon the use intended for polynucleotide sequencesencoding SDHH. For example, routine cloning, subcloning, and propagationof polynucleotide sequences encoding SDHH can be achieved using amultifunctional E. coli vector such as Bluescript® (Stratagene) orpSport1™ plasmid (GIBCO BRL). Ligation of sequences encoding SDHH intothe vector's multiple cloning site disrupts the lacZ gene, allowing acolorimetric screening procedure for identification of transformedbacteria containing recombinant molecules. In addition, these vectorsmay be useful for in vitro transcription, dideoxy sequencing, singlestrand rescue with helper phage, and creation of nested deletions in thecloned sequence. (See, e.g., Van Heeke, G. and S. M. Schuster (1989) J.Biol. Chem. 264:5503-5509.) When large quantities of SDHH are needed,e.g. for the production of antibodies, vectors which direct high levelexpression of SDHH may be used. For example, vectors containing thestrong, inducible T5 or T7 bacteriophage promoter may be used.

Yeast expression systems may be used for production of SDHH. A number ofvectors containing constitutive or inducible promoters, such as alphafactor, alcohol oxidase, and PGH, may be used in the yeast Saccharomycescerevisiae or Pichia pastoris. In addition, such vectors direct eitherthe secretion or intracellular retention of expressed proteins andenable integration of foreign sequences into the host genome for stablepropagation. (See, e.g., Ausubel, supra; and Grant et al. (1987) MethodsEnzymol. 153:516-54; Scorer, C. A. et al. (1994) Bio/Technology12:181-184.)

Plant systems may also be used for expression of SDHH. Transcription ofsequences encoding SDHH may be driven viral promoters, e.g., the 35S and19S promoters of CaMV used alone or in combination with the omega leadersequence from TMV. (Takamatsu, N. (1987) EMBO J. 6:307-311.)Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984)EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; andWinter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105.) Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. (See, e.g., Hobbs, S.or Murry, L. E. in McGraw Hill Yearbook of Science and Technology (1992)McGraw Hill, New York, N.Y.; pp. 191-196.)

In mammalian cells, a number of viral-based expression systems may beutilized. In cases where an adenovirus is used as an expression vector,sequences encoding SDHH may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain infective virus whichexpresses SDHH in host cells. (See, e.g., Logan, J. and T. Shenk (1984)Proc. Natl. Acad. Sci. 81:3655-3659.) In addition, transcriptionenhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used toincrease expression in mammalian host cells. SV40 or EBV-based vectorsmay also be used for high-level protein expression.

Human artificial chromosomes (HACs) may also be employed to deliverlarger fragments of DNA than can be contained in and expressed from aplasmid. HACs of about 6 kb to 10 Mb are constructed and delivered viaconventional delivery methods (liposomes, polycationic amino polymers,or vesicles) for therapeutic purposes.

For long term production of recombinant proteins in mammalian systems,stable expression of SDHH in cell lines is preferred. For example,sequences encoding SDHH can be transformed into cell lines usingexpression vectors which may contain viral origins of replication and/orendogenous expression elements and a selectable marker gene on the sameor on a separate vector. Following the introduction of the vector, cellsmay be allowed to grow for about 1 to 2 days in enriched media beforebeing switched to selective media. The purpose of the selectable markeris to confer resistance to a selective agent, and its presence allowsgrowth and recovery of cells which successfully express the introducedsequences. Resistant clones of stably transformed cells may bepropagated using tissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase and adenine phosphoribosyltransferase genes, for use intk⁻ or apr⁻ cells, respectively. (See, e.g., Wigler, M. et al. (1977)Cell 11:223-232; and Lowy, I. et al. (1980) Cell 22:817-823.) Also,antimetabolite, antibiotic, or herbicide resistance can be used as thebasis for selection. For example, dhfr confers resistance tomethotrexate; neo confers resistance to the aminoglycosides neomycin andG-418; and als or pat confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively. (See, e.g., Wigler, M.et al. (1980) Proc. Natl. Acad. Sci. 77:3567-3570; Colbere-Garapin, F.et al (1981) J. Mol. Biol. 150:1-14; and Murry, supra.) Additionalselectable genes have been described, e.g., trpB and hisD, which altercellular requirements for metabolites. (See, e.g., Hartman, S. C. and R.C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-8051.) Visiblemarkers, e.g., anthocyanins, green fluorescent proteins (GFP) (Clontech,Palo Alto, Calif.), β glucuronidase and its substrate β-D-glucuronoside,or luciferase and its substrate luciferin may be used. These markers canbe used not only to identify transformants, but also to quantify theamount of transient or stable protein expression attributable to aspecific vector system. (See, e.g., Rhodes, C. A. et al. (1995) MethodsMol. Biol. 55:121-131.)

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, the presence and expression of thegene may need to be confirmed. For example, if the sequence encodingSDHH is inserted within a marker gene sequence, transformed cellscontaining sequences encoding SDHH can be identified by the absence ofmarker gene function. Alternatively, a marker gene can be placed intandem with a sequence encoding SDHH under the control of a singlepromoter. Expression of the marker gene in response to induction orselection usually indicates expression of the tandem gene as well.

In general, host cells that contain the nucleic acid sequence encodingSDHH and that express SDHH may be identified by a variety of proceduresknown to those of skill in the art. These procedures include, but arenot limited to, DNA-DNA or DNA-RNA hybridizations, PCR amplification,and protein bioassay or immunoassay techniques which include membrane,solution, or chip based technologies for the detection and/orquantification of nucleic acid or protein sequences.

Immunological methods for detecting and measuring the expression of SDHHusing either specific polyclonal or monoclonal antibodies are known inthe art. Examples of such techniques include enzyme-linked immunosorbentassays (ELISAs), radioimmunoassays (RIAs), and fluorescence activatedcell sorting (FACS). A two-site, monoclonal-based immunoassay utilizingmonoclonal antibodies reactive to two non-interfering epitopes on SDHHis preferred, but a competitive binding assay may be employed. These andother assays are well known in the art. (See, e.g., Hampton, R. et al.(1990) Serological Methods, a Laboratory Manual, APS Press, St Paul,Minn., Section IV; Coligan, J. E. et al. (1997 and periodic supplements)Current Protocols in Immunology, Greene Pub. Associates andWiley-Interscience, New York, N.Y.; and Maddox, D. E. et al. (1983) J.Exp. Med. 158:1211-1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding SDHH includeoligolabeling, nick translation, end-labeling, or PCR amplificationusing a labeled nucleotide. Alternatively, the sequences encoding SDHH,or any fragments thereof, may be cloned into a vector for the productionof an mRNA probe. Such vectors are known in the art, are commerciallyavailable, and may be used to synthesize RNA probes in vitro by additionof an appropriate RNA polymerase such as T7, T3, or SP6 and labelednucleotides. These procedures may be conducted using a variety ofcommercially available kits, such as those provided by Pharmacia &Upjohn (Kalamazoo, Mich.), Promega (Madison, Wis.), and U.S. BiochemicalCorp. (Cleveland, Ohio). Suitable reporter molecules or labels which maybe used for ease of detection include radionuclides, enzymes,fluorescent, chemiluminescent, or chromogenic agents, as well assubstrates, cofactors, inhibitors, magnetic particles, and the like.

Host cells transformed with nucleotide sequences encoding SDHH may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a transformedcell may be secreted or retained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodeSDHH may be designed to contain signal sequences which direct secretionof SDHH through a prokaryotic or eukaryotic cell membrane.

In addition, a host cell strain may be chosen for its ability tomodulate expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to specify protein targeting, folding, and/oractivity. Different host cells which have specific cellular machineryand characteristic mechanisms for post-translational activities (e.g.,CHO, HeLa, MDCK, HEK293, and WI38), are available from the American TypeCulture Collection (ATCC, Bethesda, Md.) and may be chosen to ensure thecorrect modification and processing of the foreign protein.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding SDHH may be ligated to aheterologous sequence resulting in translation of a fusion protein inany of the aforementioned host systems. For example, a chimeric SDHHprotein containing a heterologous moiety that can be recognized by acommercially available antibody may facilitate the screening of peptidelibraries for inhibitors of SDHH activity. Heterologous protein andpeptide moieties may also facilitate purification of fusion proteinsusing commercially available affinity matrices. Such moieties include,but are not limited to, glutathione S-transferase (GST), maltose bindingprotein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP),6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and6-His enable purification of their cognate fusion proteins onimmobilized glutathione, maltose, phenylarsine oxide, calmodulin, andmetal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA)enable immunoaffinity purification of fusion proteins using commerciallyavailable monoclonal and polyclonal antibodies that specificallyrecognize these epitope tags. A fusion protein may also be engineered tocontain a proteolytic cleavage site located between the SDHH encodingsequence and the heterologous protein sequence, so that SDHH may becleaved away from the heterologous moiety following purification.Methods for fusion protein expression and purification are discussed inAusubel, F. M. et al. (1995 and periodic supplements) Current Protocolsin Molecular Biology, John Wiley & Sons, New York, N.Y., ch 10. Avariety of commercially available kits may also be used to facilitateexpression and purification of fusion proteins.

In a further embodiment of the invention, synthesis of radiolabeled SDHHmay be achieved in vitro using the TNT™ rabbit reticulocyte lysate orwheat germ extract systems (Promega, Madison, Wis.). These systemscouple transcription and translation of protein-coding sequencesoperably associated with the T7, T3, or SP6 promoters. Translation takesplace in the presence of a radiolabeled amino acid precursor, preferably³⁵S-methionine.

Fragments of SDHH may be produced not only by recombinant production,but also by direct peptide synthesis using solid-phase techniques. (See,e.g., Creighton, supra pp. 55-60.) Protein synthesis may be performed bymanual techniques or by automation. Automated synthesis may be achieved,for example, using the Applied Biosystems 431A Peptide Synthesizer(Perkin Elmer). Various fragments of SDHH may be synthesized separatelyand then combined to produce the full length molecule.

Therapeutics

Chemical and structural similarity exists between SDHH and rat liverserine dehydratase (GI 57225; SEQ ID NO:3), and human liver serinedehydratase (GI 338030; SEQ ID NO:4) In addition, SDHH is expressed intissues which are cancerous, proliferating, or involved in immuneresponse. Therefore, SDHH appears to play a role in disorders ofmetabolism and cancer.

In a disorder of metabolism which is associated with the activation ofdisease processes by SDHH, it is beneficial to decrease the expressionof SDHH in a subject afflicted with the disorder. In a disorder ofmetabolism which is associated with the inhibition of SDHH, it isbeneficial to provide the protein or increase the expression of SDHH ina subject afflicted with the disorder. Therefore, in one embodiment,SDHH or a fragment or derivative thereof may be administered to asubject to treat or prevent a disorder of metabolism. Such disorders caninclude, but are not limited to, Addison's disease, cystic fibrosis,diabetes, fatty hepatocirrhosis, galactosemia, goiter, hyperadrenalism,hypoadrenalism, hyperparathyroidism, hypoparathyroidism,hypercholesterolemia, hyperthyroidism, hypothyroidism hyperlipidemia,hyperlipemia, lipid myopathies, obesity, lipodystrophies,phenylketonuria, and renal failure.

In another embodiment, a vector capable of expressing SDHH or a fragmentor derivative thereof may be administered to a subject to treat orprevent a disorder of metabolism including, but not limited to, thosedescribed above.

In another embodiment, a pharmaceutical composition comprising asubstantially purified SDHH in conjunction with a suitablepharmaceutical carrier may be administered to a subject to treat orprevent a disorder of metabolism including, but not limited to, thoseprovided above.

In still another embodiment, an agonist which modulates the activity ofSDHH may be administered to a subject to treat or prevent a disorder ofmetabolism including, but not limited to, those listed above.

In a further embodiment, an antagonist of SDHH may be administered to asubject to treat or prevent a disorder of metabolism. Such a disorder ofmetabolism may include, but is not limited to, those discussed above. Inone aspect, an antibody which specifically binds SDHH may be useddirectly as an antagonist or indirectly as a targeting or deliverymechanism for bringing a pharmaceutical agent to cells or tissue whichexpress SDHH.

In an additional embodiment, a vector expressing the complement of thepolynucleotide encoding SDHH may be administered to a subject to treator prevent a a disorder of metabolism including, but not limited to,those described above.

In still another embodiment, SDHH or a fragment or derivative thereofmay be administered to a subject to treat or prevent a cancer. Suchdisorders can include, but are not limited to, adenocarcinoma, leukemia,lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, inparticular, cancers of the adrenal gland, bladder, bone, bone marrow,brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract,heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis,prostate, salivary glands, skin, spleen, testis, thymus, thyroid, anduterus.

In another embodiment, a vector capable of expressing SDHH or a fragmentor derivative thereof may be administered to a subject to treat orprevent a cancer including, but not limited to, those described above.

In a further embodiment, a pharmaceutical composition comprising asubstantially purified SDHH in conjunction with a suitablepharmaceutical carrier may be administered to a subject to treat orprevent a cancer including, but not limited to, those provided above.

In still another embodiment, an agonist which modulates the activity ofSDHH may be administered to a subject to treat or prevent a cancerincluding, but not limited to, those listed above.

In other embodiments, any of the proteins, antagonists, antibodies,agonists, complementary sequences, or vectors of the invention may beadministered in combination with other appropriate therapeutic agents.Selection of the appropriate agents for use in combination therapy maybe made by one of ordinary skill in the art, according to conventionalpharmaceutical principles. The combination of therapeutic agents may actsynergistically to effect the treatment or prevention of the variousdisorders described above. Using this approach, one may be able toachieve therapeutic efficacy with lower dosages of each agent, thusreducing the potential for adverse side effects.

An antagonist of SDHH may be produced using methods which are generallyknown in the art. In particular, purified SDHH may be used to produceantibodies or to screen libraries of pharmaceutical agents to identifythose which specifically bind SDHH. Antibodies to SDHH may also begenerated using methods that are well known in the art. Such antibodiesmay include, but are not limited to, polyclonal, monoclonal, chimeric,and single chain antibodies, Fab fragments, and fragments produced by aFab expression library. Neutralizing antibodies (i.e., those whichinhibit dimer formation) are especially preferred for therapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others may be immunized by injectionwith SDHH or with any fragment or oligopeptide thereof which hasimmunogenic properties. Depending on the host species, various adjuvantsmay be used to increase immunological response. Such adjuvants include,but are not limited to, Freund's, mineral gels such as aluminumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) andCorynebacterium parvum are especially preferable.

It is preferred that the oligopeptides, peptides, or fragments used toinduce antibodies to SDHH have an amino acid sequence consisting of atleast about 5 amino acids, and, more preferably, of at least about 10amino acids. It is also preferable that these oligopeptides, peptides,or fragments are identical to a portion of the amino acid sequence ofthe natural protein and contain the entire amino acid sequence of asmall, naturally occurring molecule. Short stretches of SDHH amino acidsmay be fused with those of another protein, such as KLH, and antibodiesto the chimeric molecule may be produced.

Monoclonal antibodies to SDHH may be prepared using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the EBV-hybridomatechnique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497;Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. etal. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; and Cole, S. P. et al.(1984) Mol. Cell Biol. 62:109-120.)

In addition, techniques developed for the production of “chimericantibodies,” such as the splicing of mouse antibody genes to humanantibody genes to obtain a molecule with appropriate antigen specificityand biological activity, can be used. (See, e.g., Morrison, S. L. et al.(1984) Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al.(1984) Nature 312:604-608; and Takeda, S. et al. (1985) Nature314:452-454.) Alternatively, techniques described for the production ofsingle chain antibodies may be adapted, using methods known in the art,to produce SDHH-specific single chain antibodies. Antibodies withrelated specificity, but of distinct idiotypic composition, may begenerated by chain shuffling from random combinatorial immunoglobulinlibraries. (See, e.g., Burton D. R. (1991) Proc. Natl. Acad. Sci.88:10134-10137.)

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature.(See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86:3833-3837; and Winter, G. et al. (1991) Nature 349:293-299.)

Antibody fragments which contain specific binding sites for SDHH mayalso be generated. For example, such fragments include, but are notlimited to, F(ab′)2 fragments produced by pepsin digestion of theantibody molecule and Fab fragments generated by reducing the disulfidebridges of the F(ab′)2 fragments. Alternatively, Fab expressionlibraries may be constructed to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity. (See, e.g., Huse,W. D. et al. (1989) Science 246:1275-1281.)

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between SDHH and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering SDHH epitopes is preferred, but a competitivebinding assay may also be employed. (Maddox, supra.)

In another embodiment of the invention, the polynucleotides encodingSDHH, or any fragment or complement thereof, may be used for therapeuticpurposes. In one aspect, the complement of the polynucleotide encodingSDHH may be used in situations in which it would be desirable to blockthe transcription of the mRNA. In particular, cells may be transformedwith sequences complementary to polynucleotides encoding SDHH. Thus,complementary molecules or fragments may be used to modulate SDHHactivity, or to achieve regulation of gene function. Such technology isnow well known in the art, and sense or antisense oligonucleotides orlarger fragments can be designed from various locations along the codingor control regions of sequences encoding SDHH.

Expression vectors derived from retroviruses, adenoviruses, or herpes orvaccinia viruses, or from various bacterial plasmids, may be used fordelivery of nucleotide sequences to the targeted organ, tissue, or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct vectors to express nucleic acid sequencescomplementary to the polynucleotides encoding SDHH. (See, e.g.,Sambrook, supra; and Ausubel, supra.)

Genes encoding SDHH can be turned off by transforming a cell or tissuewith expression vectors which express high levels of a polynucleotide,or fragment thereof, encoding SDHH. Such constructs may be used tointroduce untranslatable sense or antisense sequences into a cell. Evenin the absence of integration into the DNA, such vectors may continue totranscribe RNA molecules until they are disabled by endogenousnucleases. Transient expression may last for a month or more with anon-replicating vector, and may last even longer if appropriatereplication elements are part of the vector system.

As mentioned above, modifications of gene expression can be obtained bydesigning complementary sequences or antisense molecules (DNA, RNA, orPNA) to the control, 5′, or regulatory regions of the gene encodingSDHH. Oligonucleotides derived from the transcription initiation site,e.g., between about positions −10 and +10 from the start site, arepreferred. Similarly, inhibition can be achieved using triple helixbase-pairing methodology. Triple helix pairing is useful because itcauses inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described in the literature. (See, e.g., Gee, J. E. et al. (1994)in Huber, B. E. and B. I. Carr, Molecular and Immunologic Approaches,Futura Publishing Co., Mt. Kisco, N.Y., pp. 163-177.) A complementarysequence or antisense molecule may also be designed to block translationof mRNA by preventing the transcript from binding to ribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Forexample, engineered hammerhead motif ribozyme molecules may specificallyand efficiently catalyze endonucleolytic cleavage of sequences encodingSDHH.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites, including the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides, corresponding to the region of the target genecontaining the cleavage site, may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Complementary ribonucleic acid molecules and ribozymes of the inventionmay be prepared by any method known in the art for the synthesis ofnucleic acid molecules. These include techniques for chemicallysynthesizing oligonucleotides such as solid phase phosphoramiditechemical synthesis. Alternatively, RNA molecules may be generated by invitro and in vivo transcription of DNA sequences encoding SDHH. Such DNAsequences may be incorporated into a wide variety of vectors withsuitable RNA polymerase promoters such as T7 or SP6. Alternatively,these cDNA constructs that synthesize complementary RNA, constitutivelyor inducibly, can be introduced into cell lines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5′ and/or 3′ ends of the molecule,or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, in vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection, by liposome injections, or bypolycationic amino polymers may be achieved using methods which are wellknown in the art. (See, e.g., Goldman, C. K. et al. (1997) NatureBiotechnology 15:462-466.)

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such asdogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administrationof a pharmaceutical or sterile composition, in conjunction with apharmaceutically acceptable carrier, for any of the therapeutic effectsdiscussed above. Such pharmaceutical compositions may consist of SDHH,antibodies to SDHH, and mimetics, agonists, antagonists, or inhibitorsof SDHH. The compositions may be administered alone or in combinationwith at least one other agent, such as a stabilizing compound, which maybe administered in any sterile, biocompatible pharmaceutical carrierincluding, but not limited to, saline, buffered saline, dextrose, andwater. The compositions may be administered to a patient alone, or incombination with other agents, drugs, or hormones.

The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing Co., Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombining active compounds with solid excipient and processing theresultant mixture of granules (optionally, after grinding) to obtaintablets or dragee cores. Suitable auxiliaries can be added, if desired.Suitable excipients include carbohydrate or protein fillers, such assugars, including lactose, sucrose, mannitol, and sorbitol; starch fromcorn, wheat, rice, potato, or other plants; cellulose, such as methylcellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums, including arabic and tragacanth; andproteins, such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, and alginic acid or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with fillers or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks's solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils, such as sesame oil, or synthetic fatty acid esters, such asethyl oleate, triglycerides, or liposomes. Non-lipid polycationic aminopolymers may also be used for delivery. Optionally, the suspension mayalso contain suitable stabilizers or agents to increase the solubilityof the compounds and allow for the preparation of highly concentratedsolutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, and succinic acid. Salts tendto be more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1 mM to 50 mM histidine, 0.1% to 2% sucrose, and 2% to 7%mannitol, at a pH range of 4.5 to 5.5, that is combined with bufferprior to use.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. For administration of SDHH, such labeling would includeamount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells or inanimal models such as mice, rats, rabbits, dogs, or pigs. An animalmodel may also be used to determine the appropriate concentration rangeand route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example SDHH or fragments thereof, antibodies of SDHH,and agonists, antagonists or inhibitors of SDHH, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orwith experimental animals, such as by calculating the ED₅₀ (the dosetherapeutically effective in 50% of the population) or LD₅₀ (the doselethal to 50% of the population) statistics. The dose ratio oftherapeutic to toxic effects is the therapeutic index, and it can beexpressed as the ED₅₀/LD₅₀ ratio. Pharmaceutical compositions whichexhibit large therapeutic indices are preferred. The data obtained fromcell culture assays and animal studies are used to formulate a range ofdosage for human use. The dosage contained in such compositions ispreferably within a range of circulating concentrations that includesthe ED₅₀ with little or no toxicity. The dosage varies within this rangedepending upon the dosage form employed, the sensitivity of the patient,and the route of administration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject requiring treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, the generalhealth of the subject, the age, weight, and gender of the subject, timeand frequency of administration, drug combination(s), reactionsensitivities, and response to therapy. Long-acting pharmaceuticalcompositions may be administered every 3 to 4 days, every week, orbiweekly depending on the half-life and clearance rate of the particularformulation.

Normal dosage amounts may vary from about 0.1 μg to 100,000 μg, up to atotal dose of about 1 gram, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

DIAGNOSTICS

In another embodiment, antibodies which specifically bind SDHH may beused for the diagnosis of disorders characterized by expression of SDHH,or in assays to monitor patients being treated with SDHH or agonists,antagonists, or inhibitors of SDHH. Antibodies useful for diagnosticpurposes may be prepared in the same manner as described above fortherapeutics. Diagnostic assays for SDHH include methods which utilizethe antibody and a label to detect SDHH in human body fluids or inextracts of cells or tissues. The antibodies may be used with or withoutmodification, and may be labeled by covalent or non-covalent attachmentof a reporter molecule. A wide variety of reporter molecules, several ofwhich are described above, are known in the art and may be used.

A variety of protocols for measuring SDHH, including ELISAs, RIAs, andFACS, are known in the art and provide a basis for diagnosing altered orabnormal levels of SDHH expression. Normal or standard values for SDHHexpression are established by combining body fluids or cell extractstaken from normal mammalian subjects, preferably human, with antibody toSDHH under conditions suitable for complex formation The amount ofstandard complex formation may be quantitated by various methods,preferably by photometric means. Quantities of SDHH expressed insubject, control, and disease samples from biopsied tissues are comparedwith the standard values. Deviation between standard and subject valuesestablishes the parameters for diagnosing disease.

In another embodiment of the invention, the polynucleotides encodingSDHH may be used for diagnostic purposes. The polynucleotides which maybe used include oligonucleotide sequences, complementary RNA and DNAmolecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofSDHH may be correlated with disease. The diagnostic assay may be used todetermine absence, presence, and excess expression of SDHH, and tomonitor regulation of SDHH levels during therapeutic intervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding SDHH or closely related molecules may be used to identifynucleic acid sequences which encode SDHH. The specificity of the probe,whether it is made from a highly specific region, e.g., the 5′regulatory region, or from a less specific region, e.g., a conservedmotif, and the stringency of the hybridization or amplification(maximal, high, intermediate, or low), will determine whether the probeidentifies only naturally occurring sequences encoding SDHH, allelicvariants, or related sequences.

Probes may also be used for the detection of related sequences, andshould preferably have at least 50% sequence identity to any of the SDHHencoding sequences. The hybridization probes of the subject inventionmay be DNA or RNA and may be derived from the sequence of SEQ ID NO:2 orfrom genomic sequences including promoters, enhancers, and introns ofthe SDHH gene.

Means for producing specific hybridization probes for DNAs encoding SDHHinclude the cloning of polynucleotide sequences encoding SDHH or SDHHderivatives into vectors for the production of mRNA probes. Such vectorsare known in the art, are commercially available, and may be used tosynthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, by radionuclides such as ³²p or ³⁵S, or by enzymatic labels,such as alkaline phosphatase coupled to the probe via avidin/biotincoupling systems, and the like.

Polynucleotide sequences encoding SDHH may be used for the diagnosis ofa disorder associated with expression of SDHH. Examples of such adisorder include, but are not limited to, disorders of metabolism suchas Addison's disease, cystic fibrosis, diabetes, fatty hepatocirrhosis,galactosemia, goiter, hyperadrenalism, hypoadrenalism,hyperparathyroidism, hypoparathyroidism, hypercholesterolemia,hyperthyroidism, hypothyroidism hyperlipidemia, hyperlipemia, lipidmyopathies, obesity, lipodystrophies, phenylketonuria, and renalfailure; or a cancer such as adenocarcinoma, leukemia, lymphoma,melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancersof the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix,gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver,lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivaryglands, skin, spleen, testis, thymus, thyroid, and uterus. Thepolynucleotide sequences encoding SDHH may be used in Southern orNorthern analysis, dot blot, or other membrane-based technologies; inPCR technologies; in dipstick, pin, and ELISA assays; and in microarraysutilizing fluids or tissues from patients to detect altered SDHHexpression. Such qualitative or quantitative methods are well known inthe art.

In a particular aspect, the nucleotide sequences encoding SDHH may beuseful in assays that detect the presence of associated disorders,particularly those mentioned above. The nucleotide sequences encodingSDHH may be labeled by standard methods and added to a fluid or tissuesample from a patient under conditions suitable for the formation ofhybridization complexes. After a suitable incubation period, the sampleis washed and the signal is quantitated and compared with a standardvalue. If the amount of signal in the patient sample is significantlyaltered in comparison to a control sample then the presence of alteredlevels of nucleotide sequences encoding SDHH in the sample indicates thepresence of the associated disorder. Such assays may also be used toevaluate the efficacy of a particular therapeutic treatment regimen inanimal studies, in clinical trials, or to monitor the treatment of anindividual patient.

In order to provide a basis for the diagnosis of a disorder associatedwith expression of SDHH, a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, encoding SDHH, under conditionssuitable for hybridization or amplification. Standard hybridization maybe quantified by comparing the values obtained from normal subjects withvalues from an experiment in which a known amount of a substantiallypurified polynucleotide is used. Standard values obtained in this mannermay be compared with values obtained from samples from patients who aresymptomatic for a disorder. Deviation from standard values is used toestablish the presence of a disorder.

Once the presence of a disorder is established and a treatment protocolis initiated, hybridization assays may be repeated on a regular basis todetermine if the level of expression in the patient begins toapproximate that which is observed in the normal subject. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding SDHH may involve the use of PCR. These oligomers maybe chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably contain a fragment of a polynucleotideencoding SDHH, or a fragment of a polynucleotide complementary to thepolynucleotide encoding SDHH, and will be employed under optimizedconditions for identification of a specific gene or condition. Oligomersmay also be employed under less stringent conditions for detection orquantitation of closely related DNA or RNA sequences.

Methods which may also be used to quantitate the expression of SDHHinclude radiolabeling or biotinylating nucleotides, coamplification of acontrol nucleic acid, and interpolating results from standard curves.(See, e.g., Melby, P. C. et al. (1993) J. Immunol. Methods 159:235-244;and Duplaa, C. et al. (1993) Anal. Biochem. 229-236.) The speed ofquantitation of multiple samples may be accelerated by running the assayin an ELISA format where the oligomer of interest is presented invarious dilutions and a spectrophotometric or colorimetric responsegives rapid quantitation.

In further embodiments, oligonucleotides or longer fragments derivedfrom any of the polynucleotide sequences described herein may be used astargets in a microarray. The microarray can be used to monitor theexpression level of large numbers of genes simultaneously and toidentify genetic variants, mutations, and polymorphisms. Thisinformation may be used to determine gene function, to understand thegenetic basis of a disorder, to diagnose a disorder, and to develop andmonitor the activities of therapeutic agents.

Microarrays may be prepared, used, and analyzed using methods known inthe art. (See, e.g., Brennan, T. M. et al. (1995) U.S. Pat. No.5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci.93:10614-10619; Baldeschweiler et al. (1995) PCT application WO95/251116; Shalon, D. et al. (1995) PCT application WO95/35505; Heller, R. A.et al. (1997) Proc. Natl. Acad. Sci. 94:2150-2155; and Heller, M. J. etal. (1997) U.S. Pat. No. 5,605,662.)

In another embodiment of the invention, nucleic acid sequences encodingSDHH may be used to generate hybridization probes useful in mapping thenaturally occurring genomic sequence. The sequences may be mapped to aparticular chromosome, to a specific region of a chromosome, or toartificial chromosome constructions, e.g., human artificial chromosomes(HACs), yeast artificial chromosomes (YACs), bacterial artificialchromosomes (BACs), bacterial P1 constructions, or single chromosomecDNA libraries. (See, e.g., Price, C. M. (1993) Blood Rev. 7:127-134;and Trask, B. J. (1991) Trends Genet. 7:149-154.)

Fluorescent in situ hybridization (FISH) may be correlated with otherphysical chromosome mapping techniques and genetic map data. (See, e.g.,Heinz-Ulrich, et al. (1995) in Meyers, R. A. (ed.) Molecular Biology andBiotechnology, VCH Publishers New York, N.Y., pp. 965-968.) Examples ofgenetic map data can be found in various scientific journals or at theOnline Mendelian Inheritance in Man (OMIM) site. Correlation between thelocation of the gene encoding SDHH on a physical chromosomal map and aspecific disorder, or a predisposition to a specific disorder, may helpdefine the region of DNA associated with that disorder. The nucleotidesequences of the invention may be used to detect differences in genesequences among normal, carrier, and affected individuals.

In situ hybridization of chromosomal preparations and physical mappingtechniques, such as linkage analysis using established chromosomalmarkers, may be used for extending genetic maps. Often the placement ofa gene on the chromosome of another mammalian species, such as mouse,may reveal associated markers even if the number or arm of a particularhuman chromosome is not known. New sequences can be assigned tochromosomal arms by physical mapping. This provides valuable informationto investigators searching for disease genes using positional cloning orother gene discovery techniques. Once the disease or syndrome has beencrudely localized by genetic linkage to a particular genomic region,e.g., ataxia-telangiectasia to 11q22-23, any sequences mapping to thatarea may represent associated or regulatory genes for furtherinvestigation. (See, e.g., Gatti, R. A. et al. (1988) Nature336:577-580.) The nucleotide sequence of the subject invention may alsobe used to detect differences in the chromosomal location due totranslocation, inversion, etc., among normal, carrier, or affectedindividuals.

In another embodiment of the invention, SDHH, its catalytic orimmunogenic fragments, or oligopeptides thereof can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes between SDHHand the agent being tested may be measured.

Another technique for drug screening provides for high throughputscreening of compounds having suitable binding affinity to the proteinof interest. (See, e.g., Geysen, et al. (1984) PCT applicationWO84/03564.) In this method, large numbers of different small testcompounds are synthesized on a solid substrate, such as plastic pins orsome other surface. The test compounds are reacted with SDHH, orfragments thereof, and washed. Bound SDHH is then detected by methodswell known in the art. Purified SDHH can also be coated directly ontoplates for use in the aforementioned drug screening techniques.Alternatively, non-neutralizing antibodies can be used to capture thepeptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding SDHH specificallycompete with a test compound for binding SDHH. In this manner,antibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with SDHH.

In additional embodiments, the nucleotide sequences which encode SDHHmay be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

The examples below are provided to illustrate the subject invention andare not included for the purpose of limiting the invention.

EXAMPLES I. THP1AZS08 cDNA Library Construction

This subtracted THP-1 promonocyte cell line library was constructedusing 5.76 million clones from a 5-aza-2′-deoxycytidine (AZ) treatedTHP-1 cell library. THP-1 (ATCC TIB 202) is a human promonocyte linederived from peripheral blood of a 1-year-old Caucasian male with acutemonocytic leukemia. Starting RNA was made from THP-1 promonocyte cellstreated for three days with 0.8 micromolar AZ.

The frozen cells were homogenized and lysed using a BrinkmannHomogenizer Polytron PT-3000 (Brinkmann Instruments, Westbury, N.Y.) inguanidinium isothiocyanate solution. The lysate was centrifuged over a5.7 M CsCl cushion using an Beckman SW28 rotor in a Beckman L8-70MUltracentrifuge (Beckman Instruments) for 18 hours at 25,000 rpm atambient temperature. The mRNA was extracted with acid phenol pH 4.7,precipitated using 0.3 M sodium acetate and 2.5 volumes of ethanol,resuspended in RNAse-free water, and treated with DNase at 37° C. TheRNA was extracted and precipitated as before. The mRNA was isolated withthe Qiagen Oligotex kit (QIAGEN, Inc., Chatsworth, Calif.) and used toconstruct the THP1AZT02 cDNA library. The library was oligo(dT)-primed,and cDNAs were cloned directionally into the pSPORT1 vectoring systemusing Sal1 (5′) and NotI (3′).

The mRNA was handled according to the recommended protocols in theSuperScript Plasmid System for cDNA synthesis and plasmid cloning(Catalog #18248-013, Gibco/BRL). The cDNAs were fractionated on aSepharose CL4B column (Catalog #275105-01, Pharmacia), and those cDNAsexceeding 400 bp were ligated into pSPORT 1. The plasmid pSPORT 1 wassubsequently transformed into DH5a™ competent cells (Catalog #18258-012,Gibco/BRL).

THP1AZSO8 was constructed by subtraction of an untreated control THP1cell line library (5×10⁶ THP1NOT02 clones) from a5-aza-2′-deoxycytidine-treated THP1 cell line library. These plasmidlibraries were grown in E. coli strain DH12S (Catalog #18312-017,Gibco/BRL) liquid culture under carbenicillin (25 mg/L) and methicillin(1 mg/ml) selection following transformation by electroporation. Thecultures were checked spectrophotometrically (Model DU-7Spectrophotometer, Beckman Instruments) and allowed to grow to an OD600of 0.2, and then superinfected with a 5-fold excess of the helper phageM13K07 according to the method of Vieira et al. (Methods Enzymol. (1987)153:3-11).

To enrich for 5-aza-2′-deoxycytidine induced transcripts, the cDNAlibrary was then subtracted in two rounds of hybridization using amethodology adapted from Swaroop et al. (NAR (1991) 19:1954). TheTHP1AZT02 single-stranded library was gel and hydroxyapatite purifiedaccording to the method described in Soares et al. (Proc. Natl. Acad.Sci. (1994) 91:9228-9232.) The hybridization probe for subtraction,THP1NOT02 was generated by in vitro transcription using the AmbionMegascript kit (Catalog #1330) with SP6 RNA polymerase and 40%biotin-14-CTP (Catalog #19519-016, Gibco/BRL) following linearization ofthe double stranded plasmid DNA with Eco RI. The purifiedsingle-stranded template DNA was prehybridized according to the methodof Bonaldo et al. (Genome Research (1996) 6:791); and hybridized asdescribed in Soares, supra. In each round of subtraction, the singlestranded cDNA library derived from the 5-aza-2′-deoxycytidine-treatedcells was hybridized for 48 hours with a 300:1 molar ratio ofbiotinylated riboprobe derived from the control cell library, THP1NOT02.Following each hybridization step, the single stranded DNA (subtractedlibrary) was purified by streptavidin coated magnetic beads (Catalog#112.06, Dynal) according to the manufacturers specifications. Followingthe second streptavidin separation, the single stranded subtractedlibrary was converted to partially double-stranded by random priming,and electroporated into DH10B competent bacteria (Gibco/BRL).

II. Isolation and Sequencing of cDNA Clones

Plasmid DNA was released from the cells and purified using the REAL Prep96 Plasmid Kit for Rapid Extraction Alkaline Lysis Plasmid Minipreps(Catalog #26173, QIAGEN, Inc.). This kit enabled the simultaneouspurification of 96 samples in a 96-well block usina multichannel reagentdispensers. The recommended protocol was employed except for thefollowing changes: 1) the bacteria were cultured in 1 ml of sterileTerrific Broth (Catalog #22711, LIFE TECHNOLOGIES™) with carbenicillinat 25 mg/L and glycerol at 0.4%; 2) after inoculation, the cultures wereincubated for 19 hours and at the end of incubation, the cells werelysed with 0.3 ml of lysis buffer; and 3) following isopropanolprecipitation, the plasmid DNA pellet was resuspended in 0.1 ml ofdistilled water. After the last step in the protocol, samples weretransferred to a 96-well block for storage at 4° C.

The cDNAs were sequenced by the method of Sanger et al. (1975, J. Mol.Biol. 94:441 f), using a Hamilton Micro Lab 2200 (Hamilton, Reno, Nev.)in combination with Peltier Thermal Cyclers (PTC200 from MJ Research,Watertown, Mass.) and Applied Biosystems 377 DNA Sequencing Systems; andthe reading frame was determined.

III. Similarity Searching of cDNA Clones and Their Deduced Proteins

The nucleotide sequences and/or amino acid sequences of the SequenceListing were used to query sequences in the GenBank, SwissProt, BLOCKS,and Pima II databases. These databases, which contain previouslyidentified and annotated sequences, were searched for regions ofsimilarity using BLAST (Basic Local Alignment Search Tool). (See, e.g.,Altschul, S. F. (1993) J. Mol. Evol 36:290-300; and Altschul et al.(1990) J. Mol. Biol. 215:403-410.)

BLAST produced alignments of both nucleotide and amino acid sequences todetermine sequence similarity. Because of the local nature of thealignments, BLAST was especially useful in determining exact matches orin identifying homologs which may be of prokaryotic (bacterial) oreukaryotic (animal, fungal, or plant) origin. Other algorithms couldhave been used when dealing with primary sequence patterns and secondarystructure gap penalties. (See, e.g., Smith, T. et al. (1992) ProteinEngineering 5:35-51.) The sequences disclosed in this application havelengths of at least 49 nucleotides and have no more than 12% uncalledbases (where N is recorded rather than A, C, G, or T).

The BLAST approach searched for matches between a query sequence and adatabase sequence. BLAST evaluated the statistical significance of anymatches found, and reported only those matches that satisfy theuser-selected threshold of significance. In this application, thresholdwas set at 10⁻²⁵ for nucleotides and 10⁻⁸ for peptides.

Incyte nucleotide sequences were searched against the GenBank databasesfor primate (pri), rodent (rod), and other mammalian sequences (mam),and deduced amino acid sequences from the same clones were then searchedagainst GenBank functional protein databases, mammalian (mamp),vertebrate (vrtp), and eukaryote (eukp), for similarity.

Additionally, sequences identified from cDNA libraries may be analyzedto identify those gene sequences encoding conserved protein motifs usingan appropriate analysis program, e.g., BLOCKS. BLOCKS is a weightedmatrix analysis algorithm based on short amino acid segments, or blocks,compiled from the PROSITE database. (Bairoch, A. et al. (1997) NucleicAcids Res. 25:217-221.) The BLOCKS algorithm is useful for classifyinggenes with unknown functions. (Henikoff S. And Henikoff G. J., NucleicAcids Research (1991) 19:6565-6572.) Blocks, which are 3-60 amino acidsin length, correspond to the most highly conserved regions of proteins.The BLOCKS algorithm compares a query sequence with a weighted scoringmatrix of blocks in the BLOCKS database. Blocks in the BLOCKS databaseare calibrated against protein sequences with known functions from theSWISS-PROT database to determine the stochastic distribution of matches.Similar databases such as PRINTS, a protein fingerprint database, arealso searchable using the BLOCKS algorithm. (Attwood, T. K. et al.(1997) J. Chem. Inf. Comput. Sci. 37:417-424.) PRINTS is based onnon-redundant sequences obtained from sources such as SWISS-PROT,GenBank, PIR, and NRL-3D.

The BLOCKS algorithm searches for matches between a query sequence andthe BLOCKS or PRINTS database and evaluates the statistical significanceof any matches found. Matches from a BLOCKS or PRINTS search can beevaluated on two levels, local similarity and global similarity. Thedegree of local similarity is measured by scores, and the extent ofglobal similarity is measured by score ranking and probability values. Ascore of 1000 or greater for a BLOCKS match of highest ranking indicatesthat the match falls within the 0.5 percentile level of false positiveswhen the matched block is calibrated against SWISS-PROT. Likewise, aprobability value of less than 1.0×10⁻³ indicates that the match wouldoccur by chance no more than one time in every 1000 searches. Only thosematches with a cutoff score of 1000 or greater and a cutoff probabilityvalue of 1.0×10⁻³ or less are considered in the functional analyses ofthe protein sequences in the Sequence Listing.

Nucleic and amino acid sequences of the Sequence Listing may also beanalyzed using PFAM. PFAM is a Hidden Markov Model (HMM) based protocoluseful in protein family searching. HMM is a probabilistic approachwhich analyzes consensus primary structures of gene families. (See,e.g., Eddy, S. R. (1996) Cur. Opin. Str. Biol. 6:361-365.)

The PFAM database contains protein sequences of 527 protein familiesgathered from publicly available sources, e.g., SWISS-PROT and PROSITE.PFAM searches for well characterized protein domain families using twohigh-quality alignment routines, seed alignment and full alignment.(See, e.g., Sonnhammer, E. L. L. et al. (1997) Proteins 28:405-420.) Theseed alignment utilizes the hmmls program, a program that searches forlocal matches, and a non-redundant set of the PFAM database. The fullalignment utilizes the hmmfs program, a program that searches formultiple fragments in long sequences, e.g., repeats and motifs, and allsequences in the PFAM database. A result or score of 100 “bits” cansignify that it is 2¹⁰⁰-fold more likely that the sequence is a truematch to the model or comparison sequence. Cutoff scores which rangefrom 10 to 50 bits are generally used for individual protein familiesusing the SWISS-PROT sequences as model or comparison sequences.

Two other algorithms, SIGPEPT and TM, both based on the HMM algorithmdescribed above (see, e.g., Eddy, supra; and Sonnhammer, supra),identify potential signal sequences and transmembrane domains,respectively. SIGPEPT was created using protein sequences having signalsequence annotations derived from SWISS-PROT. It contains about 1413non-redundant signal sequences ranging in length from 14 to 36 aminoacid residues. TM was created similarly using transmembrane domainannotations. It contains about 453 non-redundant transmembrane sequencesencompassing 1579 transmembrane domain segments. Suitable HMM modelswere constructed using the above sequences and were refined with knownSWISS-PROT signal peptide sequences or transmembrane domain sequencesuntil a high correlation coefficient, a measurement of the correctnessof the analysis, was obtained. Using the protein sequences from theSWISS-PROT database as a test set, a cutoff score of 11 bits, asdetermined above, correlated with 91-94% true-positives and about 4.1%false-positives, yielding a correlation coefficient of about 0.87-0.90for SIGPEPT. A score of 11 bits for TM will typically give the followingresults: 75% true positives; 1.72% false positives; and a correlationcoefficient of 0.76. Each search evaluates the statistical significanceof any matches found and reports only those matches that score at least11 bits.

IV. Northern Analysis

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound. (See, e.g., Sambrook, supra, ch. 7; andAusubel, supra, ch. 4 and 16.)

Analogous computer techniques applying BLAST are used to search foridentical or related molecules in nucleotide databases such as GenBankor LIFESEQ™ database (Incyte Pharmaceuticals). This analysis is muchfaster than multiple membrane-based hybridizations. In addition, thesensitivity of the computer search can be modified to determine whetherany particular match is categorized as exact or similar.

The basis of the search is the product score, which is defined as:$\frac{\% \quad {sequence}\quad {identity} \times \% \quad {maximum}\quad {BLAST}\quad {score}}{100}$

The product score takes into account both the degree of similaritybetween two sequences and the length of the sequence match. For example,with a product score of 40, the match will be exact within a 1% to 2%error, and, with a product score of 70, the match will be exact. Similarmolecules are usually identified by selecting those which show productscores between 15 and 40, although lower scores may identify relatedmolecules.

The results of Northern analysis are reported as a list of libraries inwhich the transcript encoding SDHH occurs. Abundance and percentabundance are also reported. Abundance directly reflects the number oftimes a particular transcript is represented in a cDNA library, andpercent abundance is abundance divided by the total number of sequencesexamined in the cDNA library.

V. Extension of SDHH Encoding Polynucleotides

The nucleic acid sequence of Incyte Clone 2752518 was used to designoligonucleotide primers for extending a partial nucleotide sequence tofull length. One primer was synthesized to initiate extension of anantisense polynucleotide, and the other was synthesized to initiateextension of a sense polynucleotide. Primers were used to facilitate theextension of the known sequence “outward” generating ampliconscontaining new unknown nucleotide sequence for the region of interest.The initial primers were designed from the cDNA using OLIGO™ 4.06(National Biosciences, Plymouth, Minn.), or another appropriate program,to be about 22 to 30 nucleotides in length, to have a GC content ofabout 50% or more, and to anneal to the target sequence at temperaturesof about 68° C. to about 72° C. Any stretch of nucleotides which wouldresult in hairpin structures and primer-primer dimerizations wasavoided.

Selected human cDNA libraries (GIBCO BRL) were used to extend thesequence. If more than one extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

High fidelity amplification was obtained by following the instructionsfor the XL-PCR™ kit (Perkin Elmer) and thoroughly mixing the enzyme andreaction mix. PCR was performed using the Peltier Thermal Cycler(PTC200; M.J. Research, Watertown, Mass.), beginning with 40 pmol ofeach primer and the recommended concentrations of all other componentsof the kit, with the following parameters:

Step 1 94° C. for 1 min (initial denaturation) Step 2 65° C. for 1 minStep 3 68° C. for 6 min Step 4 94° C. for 15 sec Step 5 65° C. for 1 minStep 6 68° C. for 7 min Step 7 Repeat steps 4 through 6 for anadditional 15 cycles Step 8 94° C. for 15 sec Step 9 65° C. for 1 minStep 10 68° C. for 7:15 min Step 11 Repeat steps 8 through 10 for anadditional 12 cycles Step 12 72° C. for 8 min Step 13 4° C. (andholding)

A 5 μl to 10 μl aliquot of the reaction mixture was analyzed byelectrophoresis on a low concentration (about 0.6% to 0.8%) agarosemini-gel to determine which reactions were successful in extending thesequence. Bands thought to contain the largest products were excisedfrom the gel, purified using QIAQUICK™ (QIAGEN Inc.), and trimmed ofoverhangs using Klenow enzyme to facilitate religation and cloning.

After ethanol precipitation, the products were redissolved in 13 μl ofligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase were added, and the mixture was incubated at roomtemperature for 2 to 3 hours, or overnight at 16° C. Competent E. colicells (in 40 μl of appropriate media) were transformed with 3 μl ofligation mixture and cultured in 80 μl of SOC medium. (See, e.g.,Sambrook, supra, Appendix A, p. 2.) After incubation for one hour at 37°C., the E. coli mixture was plated on Luria Bertani (LB) agar (See,e.g., Sambrook, supra, Appendix A, p. 1) containing carbenicillin(2×carb). The following day, several colonies were randomly picked fromeach plate and cultured in 150 μl of liquid LB/2×carb medium placed inan individual well of an appropriate commercially-available sterile96-well microtiter plate. The following day, 5 μl of each overnightculture was transferred into a non-sterile 96-well plate and, afterdilution 1:10 with water, 5 μl from each sample was transferred into aPCR array.

For PCR amplification, 18 μl of concentrated PCR reaction mix (3.3×)containing 4 units of rTth DNA polymerase, a vector primer, and one orboth of the gene specific primers used for the extension reaction wereadded to each well. Amplification was performed using the followingconditions:

Step 1 94° C. for 60 sec Step 2 94° C. for 20 sec Step 3 55° C. for 30sec Step 4 72° C. for 90 sec Step 5 Repeat steps 2 through 4 for anadditional 29 cycles Step 6 72° C. for 180 sec Step 7 4° C. (andholding)

Aliquots of the PCR reactions were run on agarose gels together withmolecular weight markers. The sizes of the PCR products were compared tothe original partial cDNAs, and appropriate clones were selected,ligated into plasmid, and sequenced.

In like manner, the nucleotide sequence of SEQ ID NO:2 is used to obtain5′ regulatory sequences using the procedure above, oligonucleotidesdesigned for 5′ extension, and an appropriate genomic library.

VI. Labeling and Use of Individual Hybridization Probes

Hybridization probes derived from SEQ ID NO:2 are employed to screencDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base pairs, is specificallydescribed, essentially the same procedure is used with larger nucleotidefragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO™ 4.06 software (National Biosciences) and labeled bycombining 50 pmol of each oligomer, 250 μCi of [y-³²P] adenosinetriphosphate (Amersham, Chicago, Ill.), and T4 polynucleotide kinase(DuPont NEN®, Boston, Mass.). The labeled oligonucleotides aresubstantially purified using a Sephadex™ G-25 superfine size exclusiondextran bead column (Pharmacia & Upjohn, Kalamazoo, Mich.). An aliquotcontaining 10⁷ counts per minute of the labeled probe is used in atypical membrane-based hybridization analysis of human genomic DNAdigested with one of the following endonucleases: Ase I, Bgl II, Eco RI,Pst I, Xba1, or Pvu II (DuPont NEN, Boston, Mass.).

The DNA from each digest is fractionated on a 0.7% agarose gel andtransferred to nylon membranes (Nytran Plus, Schleicher & Schuell,Durham, N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1×salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR™ film(Kodak, Rochester, N.Y.) is exposed to the blots to film for severalhours, hybridization patterns are compared visually.

VII. Microarrays

A chemical coupling procedure and an ink jet device can be used tosynthesize array elements on the surface of a substrate. (See, e.g.,Baldeschweiler, supra.) An array analogous to a dot or slot blot mayalso be used to arrange and link elements to the surface of a substrateusing thermal, UV, chemical, or mechanical bonding procedures. A typicalarray may be produced by hand or using available methods and machinesand contain any appropriate number of elements. After hybridization,nonhybridized probes are removed and a scanner used to determine thelevels and patterns of fluorescence. The degree of complementarity andthe relative abundance of each probe which hybridizes to an element onthe microarray may be assessed through analysis of the scanned images.

Full-length cDNAs, Expressed Sequence Tags (ESTs), or fragments thereofmay comprise the elements of the microarray. Fragments suitable forhybridization can be selected using software well known in the art suchas LASERGENE™. Full-length cDNAs, ESTs, or fragments thereofcorresponding to one of the nucleotide sequences of the presentinvention, or selected at random from a cDNA library relevant to thepresent invention, are arranged on an appropriate substrate, e.g., aglass slide. The cDNA is fixed to the slide using, e.g., UVcross-linking followed by thermal and chemical treatments and subsequentdrying. (See, e.g., Schena, M. et al. (1995) Science 270:467-470; andShalon, D. et al. (1996) Genome Res. 6:639-645.) Fluorescent probes areprepared and used for hybridization to the elements on the substrate.The substrate is analyzed by procedures described above.

VIII. Complementary Polynucleotides

Sequences complementary to the SDHH-encoding sequences, or any partsthereof, are used to detect, decrease, or inhibit expression ofnaturally occurring SDHH. Although use of oligonucleotides comprisingfrom about 15 to 30 base pairs is described, essentially the sameprocedure is used with smaller or with larger sequence fragments.Appropriate oligonucleotides are designed using OLIGO™ 4.06 software andthe coding sequence of SDHH. To inhibit transcription, a complementaryoligonucleotide is designed from the most unique 5′ sequence and used toprevent promoter binding to the coding sequence. To inhibit translation,a complementary oligonucleotide is designed to prevent ribosomal bindingto the SDHH-encoding transcript.

X. Expression of SDHH

Expression and purification of SDHH is achieved using bacterial orvirus-based expression systems. For expression of SDHH in bacteria, cDNAis subcloned into an appropriate vector containing an antibioticresistance gene and an inducible promoter that directs high levels ofcDNA transcription. Examples of such promoters include, but are notlimited to, the trp-lac (tac) hybrid promoter and the T5 or T7bacteriophage promoter in conjunction with the lac operator regulatoryelement. Recombinant vectors are transformed into suitable bacterialhosts, e.g., BL21(DE3). Antibiotic resistant bacteria express SDHH uponinduction with isopropyl beta-D-thiogalactopyranoside (IPTG). Expressionof SDHH in eukaryotic cells is achieved by infecting insect or mammaliancell lines with recombinant Autographica californica nuclearpolyhedrosis virus (AcMNPV), commonly known as baculovirus. Thenonessential polyhedrin gene of baculovirus is replaced with cDNAencoding SDHH by either homologous recombination or bacterial-mediatedtransposition involving transfer plasmid intermediates. Viralinfectivity is maintained and the strong polyhedrin promoter drives highlevels of cDNA transcription. Recombinant baculovirus is used to infectSpodoptera frugiperda (Sf9) insect cells in most cases, or humanhepatocytes, in some cases. Infection of the latter requires additionalgenetic modifications to baculovirus. (See Engelhard, E. K. et al.(1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996)Hum. Gene Ther. 7:1937-1945.)

In most expression systems, SDHH is synthesized as a fusion proteinwith, e.g., glutathione S-transferase (GST) or a peptide epitope tag,such as FLAG or 6-His, permitting rapid, single-step, affinity-basedpurification of recombinant fusion protein from crude cell lysates. GST,a 26-kilodalton enzyme from Schistosoma japonicum, enables thepurification of fusion proteins on immobilized glutathione underconditions that maintain protein activity and antigenicity (Pharmacia,Piscataway, N.J.). Following purification, the GST moiety can beproteolytically cleaved from SDHH at specifically engineered sites.FLAG, an 8-amino acid peptide, enables immunoaffinity purification usingcommercially available monoclonal and polyclonal anti-FLAG antibodies(Eastman Kodak, Rochester, N.Y.). 6-His, a stretch of six consecutivehistidine residues, enables purification on metal-chelate resins (QIAGENInc, Chatsworth, Calif.). Methods for protein expression andpurification are discussed in Ausubel, F. M. et al. (1995 and periodicsupplements) Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y., ch 10, 16. Purified SDHH obtained by these methods canbe used directly in the following activity assay.

X. Demonstration of SDHH Activity

SDHH activity may be conveniently determined by measuring the conversionof serine to pyruvate. (Suda et al. (1970) Meth. Enzymol.17B:346-351). Asolution of 1 M serine in 0.1 M potassium phosphate buffer, pH 8.0 isprepared and stored at −20° C. The enzyme is combined with 0.1 ml 5×10⁻⁴M pyridoxal phosphate, 0.5 ml 0.2 M phosphate buffer at pH 8 containing2×10⁻³ M EDTA, the volume is adjusted to 0.9 ml with water, and thesolution is warmed in a 37° C. water bath for 5 minutes. 0.1 ml of theserine solution warmed to 37° C. is added and incubated for 5 minutes at37° C. The reaction is stopped with 0.5 ml of 10% trichloroacetic acid.The preparation kept in an ice bath for 10 minutes. Any precipitate isremoved by centrifugation. A 0.5 ml aliquot is combined with 0.5 ml0.033% 2,4-dinitrophenylhydrazine solution in 2N HCl and the mixture isallowed to stand for at least 5 minutes at approximately 20°. Anypyruvate formed is converted to the hydrazine in the presence of2,4-dinitrophenylhydrazine. 2 ml of 2N sodium hydroxide solution isadded to stop the reaction. The absorbance of the reaction mixture isread at 520 nm within 5 minutes, using a spectrophotometer. Absorbanceat this frequency is proportional to the SDHH activity in the originalsample. For a control, trichloroacetic acid is added to the reactionmixture prior to the addition of the substrate.

XI. Functional Assays

SDHH function is assessed by expressing the sequences encoding SDHH atphysiologically elevated levels in mammalian cell culture systems. cDNAis subcloned into a mammalian expression vector containing a strongpromoter that drives high levels of cDNA expression. Vectors of choiceinclude pCMV SPORT™ (Life Technologies, Gaithersburg, Md.) and pCR™ 3.1(Invitrogen, Carlsbad, Calif., both of which contain the cytomegaloviruspromoter. 5-10 μg of recombinant vector are transiently transfected intoa human cell line, preferably of endothelial or hematopoietic origin,using either liposome formulations or electroporation. 1-2 μg of anadditional plasmid containing sequences encoding a marker protein areco-transfected. Expression of a marker protein provides a means todistinguish transfected cells from nontransfected cells and is areliable predictor of cDNA expression from the recombinant vector.Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP)(Clontech, Palo Alto, Calif.), CD64, or a CD64-GFP fusion protein. Flowcytometry (FCM), an automated, laser optics-based technique, is used toidentify transfected cells expressing GFP or CD64-GFP, and to evaluateproperties, for example, their apoptotic state. FCM detects andquantifies the uptake of fluorescent molecules that diagnose eventspreceding or coincident with cell death. These events include changes innuclear DNA content as measured by staining of DNA with propidiumiodide; changes in cell size and granularity as measured by forwardlight scatter and 90 degree side light scatter; down-regulation of DNAsynthesis as measured by decrease in bromodeoxyuridine uptake;alterations in expression of cell surface and intracellular proteins asmeasured by reactivity with specific antibodies; and alterations inplasma membrane composition as measured by the binding offluorescein-conjugated Annexin V protein to the cell surface. Methods inflow cytometry are discussed in Ormerod, M. G. (1994) Flow Cytometry,Oxford, New York, N.Y.

The influence of SDHH on gene expression can be assessed using highlypurified populations of cells transfected with sequences encoding SDHHand either CD64 or CD64-GFP. CD64 and CD64-GFP are expressed on thesurface of transfected cells and bind to conserved regions of humanimmunoglobulin G (IgG). Transfected cells are efficiently separated fromnontransfected cells using magnetic beads coated with either human IgGor antibody against CD64 (DYNAL, Lake Success, N.Y.). mRNA can bepurified from the cells using methods well known by those of skill inthe art. Expression of mRNA encoding SDHH and other genes of interestcan be analyzed by Northern analysis or microarray techniques.

XII. Production of SDHH Specific Antibodies

SDHH substantially purified using polyacrylamide gel electrophoresis(PAGE)(see, e.g., Harrington, M. G. (1990) Methods Enzymol.182:488-495), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols.

Alternatively, the SDHH amino acid sequence is analyzed using LASERGENE™software (DNASTAR Inc.) to determine regions of high immunogenicity, anda corresponding oligopeptide is synthesized and used to raise antibodiesby means known to those of skill in the art. Methods for selection ofappropriate epitopes, such as those near the C-terminus or inhydrophilic regions are well described in the art. (See, e.g., Ausubelsupra, ch. 11.)

Typically, oligopeptides 15 residues in length are synthesized using anApplied Biosystems Peptide Synthesizer Model 431 A using fmoc-chemistryand coupled to KLH (Sigma, St. Louis, Mo.) by reaction withN-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increaseimmunogenicity. (See, e.g., Ausubel supra.) Rabbits are immunized withthe oligopeptide-KLH complex in complete Freund's adjuvant. Resultingantisera are tested for antipeptide activity by, for example, bindingthe peptide to plastic, blocking with 1% BSA, reacting with rabbitantisera, washing, and reacting with radio-iodinated goat anti-rabbitIgG.

XIII. Purification of Naturally Occurring SDHH Using Specific Antibodies

Naturally occurring or recombinant SDHH is substantially purified byimmunoaffinity chromatography using antibodies specific for SDHH. Animmunoaffinity column is constructed by covalently coupling anti-SDHHantibody to an activated chromatographic resin, such as CNBr-activatedSepharose (Pharmacia & Upjohn). After the coupling, the resin is blockedand washed according to the manufacturer's instructions.

Media containing SDHH are passed over the immunoaffinity column, and thecolumn is washed under conditions that allow the preferential absorbanceof SDHH (e.g., high ionic strength buffers in the presence ofdetergent). The column is eluted under conditions that disruptantibody/SDHH binding (e.g., a buffer of pH 2 to pH 3, or a highconcentration of a chaotrope, such as urea or thiocyanate ion), and SDHHis collected.

XIV. Identification of Molecules Which Interact with SDHH

SDHH, or biologically active fragments thereof, are labeled with ¹²⁵IBolton-Hunter reagent. (See, e.g., Bolton et al. (1973) Biochem. J.133:529.) Candidate molecules previously arrayed in the wells of amulti-well plate are incubated with the labeled SDHH, washed, and anywells with labeled SDHH complex are assayed. Data obtained usingdifferent concentrations of SDHH are used to calculate values for thenumber, affinity, and association of SDHH with the candidate molecules.

Various modifications and variations of the described methods andsystems of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughthe invention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the followingclaims.

2 325 amino acids amino acid single linear not provided THP1AZS082752518 1 Met Asp Gly Pro Val Ala Glu His Ala Lys Gln Glu Pro Phe HisVal 1 5 10 15 Val Thr Pro Leu Leu Glu Ser Trp Ala Leu Ser Gln Val AlaGly Met 20 25 30 Pro Val Phe Leu Lys Cys Glu Asn Val Gln Pro Ser Gly SerPhe Lys 35 40 45 Ile Arg Gly Ile Gly His Phe Cys Gln Glu Met Ala Lys LysGly Cys 50 55 60 Arg His Leu Val Cys Ser Ser Gly Gly Asn Ala Gly Ile AlaAla Ala 65 70 75 80 Tyr Ala Ala Arg Lys Leu Gly Ile Pro Ala Thr Ile ValLeu Pro Glu 85 90 95 Ser Thr Ser Leu Gln Val Val Gln Arg Leu Gln Gly GluGly Ala Glu 100 105 110 Val Gln Leu Thr Gly Lys Val Trp Asp Glu Ala AsnLeu Arg Ala Gln 115 120 125 Glu Leu Ala Lys Arg Asp Gly Trp Glu Asn ValPro Pro Phe Asp His 130 135 140 Pro Leu Ile Trp Lys Gly His Ala Ser LeuVal Gln Glu Leu Lys Ala 145 150 155 160 Val Leu Arg Thr Pro Pro Gly AlaLeu Val Leu Ala Val Gly Gly Gly 165 170 175 Gly Leu Leu Ala Gly Val ValAla Gly Leu Leu Glu Val Gly Trp Gln 180 185 190 His Val Pro Ile Ile AlaMet His Gly Ala His Cys Phe Asn Ala Ala 195 200 205 Ile Thr Ala Gly LysLeu Val Thr Leu Pro Asp Ile Thr Ser Val Ala 210 215 220 Lys Ser Leu GlyAla Lys Thr Val Ala Ala Arg Ala Leu Glu Cys Met 225 230 235 240 Gln ValCys Lys Ile His Ser Glu Val Val Glu Asp Thr Glu Ala Val 245 250 255 SerAla Val Gln Gln Leu Leu Asp Asp Glu Arg Met Leu Val Glu Pro 260 265 270Ala Cys Gly Ala Ala Ala Ile Tyr Ser Gly Leu Leu Arg Arg Leu Gln 275 280285 Ala Glu Gly Cys Leu Pro Pro Ser Leu Thr Ser Val Val Val Ile Val 290295 300 Cys Gly Gly Asn Asn Ile Asn Ser Arg Glu Leu Gln Ala Leu Lys Thr305 310 315 320 His Leu Gly Gln Val 325 1485 base pairs nucleic acidsingle linear not provided THP1AZS08 2752518 2 GGGTCGACCA CCGTCGCATGATTAGGATGG TGCATGAGTG ATCGGCAGTG CCCGGGAAAG 60 CGGTGAGGGT TGCTCTCATCCCCTCTCCTC CTCCGTCTTC ACCCGGAGGC TTAGGGTCTG 120 GAGCTTTCTC TTTAACAAAGGAGGAGGGAC CAAGGTTGCC GGAAGCTGCC TGAAGCTGGA 180 CAGAGCCGGT TCCTGGAAAGAGCTGGTTCC CTGGCAGGCT GGAGGGCAGG AGCTGGGGCC 240 ACGCTGGTCT GGGATAGTTGGGCAGGGAGG CTGTCTACCT GGTCTCCAGA ATGGACGGCC 300 CTGTGGCAGA GCATGCCAAGCAGGAGCCCT TTCACGTGGT CACACCTCTG TTGGAGAGCT 360 GGGCGCTGTC CCAGGTGGCGGGCATGCCTG TCTTCCTCAA GTGTGAGAAT GTGCAGCCCA 420 GCGGCTCCTT CAAGATTCGGGGCATTGGGC ATTTCTGCCA GGAGATGGCC AAGAAGGGAT 480 GCAGACACCT GGTGTGCTCCTCAGGGGGTA ATGCGGGCAT CGCTGCTGCC TATGCTGCTA 540 GGAAGCTGGG CATTCCTGCCACCATCGTGC TCCCCGAGAG CACCTCCCTG CAGGTGGTGC 600 AGAGGCTGCA GGGGGAGGGGGCCGAGGTTC AGCTGACTGG AAAGGTCTGG GACGAGGCCA 660 ATCTGAGGGC GCAAGAGTTGGCCAAGAGGG ACGGCTGGGA GAATGTCCCC CCGTTTGACC 720 ACCCCCTAAT ATGGAAAGGCCACGCCAGCC TGGTGCAGGA GCTGAAAGCA GTGCTGAGGA 780 CCCCACCAGG TGCCCTGGTGCTGGCAGTTG GGGGTGGGGG TCTCCTGGCC GGGGTGGTGG 840 CTGGCCTGCT GGAGGTGGGCTGGCAGCATG TACCCATCAT TGCCATGGAG ACCCATGGGG 900 CACACTGCTT CAATGCGGCCATCACAGCCG GCAAGCTGGT CACACTTCCA GACATCACCA 960 GTGTGGCCAA GAGCCTGGGTGCCAAGACGG TGGCCGCTCG GGCCCTGGAG TGCATGCAGG 1020 TGTGCAAGAT TCACTCTGAAGTGGTGGAGG ACACCGAGGC TGTGAGCGCT GTGCAGCAGC 1080 TCCTGGATGA TGAGCGTATGCTGGTGGAGC CTGCCTGTGG GGCAGCCTTA GCAGCCATCT 1140 ACTCAGGCCT CCTGCGGAGGCTCCAGGCCG AGGGCTGCCT GCCCCCTTCC CTGACTTCAG 1200 TTGTGGTAAT CGTGTGTGGAGGCAACAACA TCAACAGCCG AGAGCTGCAG GCTTTGAAAA 1260 CCCACCTGGG CCAGGTCTGAGGGGTCCCAT CCTGGCCCCA AAGACCCCTG AGAGGCCCAT 1320 GGACAGTCCT GTGTCTGGATGAGGAGGACT CAGTGCTGGC AGATGGCAGT GGAAGCTGCC 1380 CTGTGCAACT GTGCTGGCTGCCTCCTGAAG GAAGCCCTCC TGGACTGCTT CTTTTGGCTC 1440 TCCGACAACT CCGGCCAATAAACACTTTCT GAATTGAAAA AAAAA 1485

What is claimed is:
 1. An isolated purified polypeptide comprising anamino acid sequence selected from the group consisting of: a) an aminoacid sequence of SEQ ID NO:1, b) an amino acid sequence having at least90% sequence identity to the amino acid sequence of SEQ ID NO:1, whereinsaid amino acid sequence has SDHH activity, c) a biologically activefragment of the amino acid sequence of SEQ ID NO:1, wherein said aminoacid sequence has SDHH activity, and d) an immunogenic fragment of theamino acid sequence of SEQ ID NO:1, wherein said fragment generates anantibody that specifically binds to the polypeptide having the aminoacid sequence of SEQ ID NO:1.
 2. An isolated polypeptide of claim 1,having an amino acid sequence of SEQ ID NO:1.
 3. A compositioncomprising a polypeptide of claim 1 and a pharmaceutically acceptableexcipient.